p63 inactivation for the treatment of heart failure

ABSTRACT

Embodiments of the disclosure include methods and compositions for in situ cardiac cell regeneration, including transdifferentiation of cardiac cells to cardiomyocytes. In particular embodiments, in situ cardiac cell regeneration encompasses delivery of p63 shRNA and one or both of Hand2 and myocardin, and in specific embodiments further includes one or more of Gata4, Mef2c, and Tbx5. In specific aspects of the disclosure, adult cardiac fibroblasts are reprogrammed into cardiomyocytes using viral vectors that harbor p63 shRNA and one or both of the transcription factors Hand2 and myocardin.

This application is a national phase application under 35 U.S.C. § 371that claims priority to International Application No. PCT/US2016/018735filed Feb. 19, 2016 which claims the benefit of priority to U.S.Provisional Patent Application Ser. No. 62/118,573, filed Feb. 20, 2015,all of which are incorporated by reference herein in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under HL121294 awardedby the National Institutes of Health. The government has certain rightsin the invention.

TECHNICAL FIELD

Embodiments of the disclosure include at least the fields of molecularbiology, cell biology, cell therapy, and medicine, including cardiacmedicine.

BACKGROUND OF THE INVENTION

Despite medical and surgical innovations, heart disease remains thenumber one cause of death in the world. Given the poor regenerativecapacity of the heart following myocardial infarction and theirreversible loss of cardiomyocytes, the replacement of cardiomyocytesby forming induced pluripotent stem cells or stimulating direct cellularreprogramming are potential therapeutic strategies that hold greatpromise. Both technologies are rooted in the idea that endogenousfibroblasts within the infarcted myocardium can be reprogrammed intofunctional cardiomyocytes. Several institutions have reported that acocktail of transcription factors, most notably Gata4, Mef2c, and Tbx5,can be used to reprogram fibroblasts into cardiomyocytes in vitro.Nevertheless, the major obstacle in the implementation of this therapyis the low efficiency of reprogramming.

The present disclosure provides solutions to a long-felt need in the artfor efficient and effective repair of cardiac tissue.

BRIEF SUMMARY OF THE INVENTION

Embodiments of the disclosure include methods and compositions for thetreatment of any medical condition related to the mammalian heart. Inspecific embodiments, the disclosure concerns treatment of one or morecardiac medical conditions with therapeutic compositions that affectendogenous cells or tissue in the heart. In particular embodiments,therapy is provided to an individual in need thereof, such as when theindividual has a need for in situ or in vivo therapy of endogenouscardiac tissue because of a cardiac medical condition or risk thereof.In specific embodiments, the individual has cardiac cellular or cardiactissue damage from a cardiac medical condition.

In particular embodiments, delivery of certain composition(s) to cellsin situ or in vivo in the individual allows regeneration ofcardiomyocytes by allowing reprogramming of endogenous non-cardiomyocytecells to become cardiomyocytes. Upon delivery of a therapeuticallyeffective amount of one or more composition(s) to the individual, thecomposition(s) provide improvement of the condition at least in part,such as by allowing regeneration of cardiac tissue or cells therein. Inspecific embodiments, the composition(s) comprise one or more moleculesfor inactivation of p63, p53, p21, p16, p19, p38, and/or p57 (such as byat least one kind of RNA interference), or other factors thedownregulation of which would enhance the reprogrammability or“plasticity of target cells. In certain embodiments, an individual mayalso be provided with one or more cardiac cell reprogramming factors(which may or may not be transcription factors). In particularembodiments, the composition(s) comprise Hand2, myocardin, or both. Inspecific embodiments, the RNA interference molecule for p63 inactivationcomprises small interfering RNA (siRNA), short hairpin RNA (shRNA) orbi-functional shRNA, as examples. In certain embodiments, an individualis also provided with one or more chromatin destabilizing agents.

In particular embodiments of the disclosure, p63, p53, and/or p21inactivation increases the transdifferentiation efficiency of cardiaccells (such as fibroblasts) into cardiomyocytes. As described herein,the partial or complete inactivation of the p63, p53, and/or p21 genesis a novel therapeutic intervention that allows the reprogramming offibroblasts (for example) into cardiomyocytes at a much higherefficiency. In vitro studies confirm that 45% of p63 knockout mouseembryonic fibroblasts express cardiac Troponin T (cTnT), which is ahighly specific marker for the cardiomyocyte lineage. p63 inactivationthrough shRNA or siRNA (for example) is a unique intervention that is aclinically relevant therapy for the treatment of any cardiac medicalcondition, including heart failure.

In specific embodiments, any p63, p53, and/or p21-inactivation agentand/or one or more cardiac cell reprogramming factors and/or one or morechromatin destabilizing agents and/or one or more anti-fibrotic agentsand/or one or more angiogenic factors act synergistically with eachother.

In specific embodiments, an individual in need thereof receives one ormore anti-fibrotic agents, such as one or more anti-Snail agents (forexample, siRNA, antibody, small molecule such as ITD-1, etc.).

in one embodiment, there is a method of in vivo reprogramming of cardiaccells, comprising the step of providing a therapeutically effectiveamount of one or more compositions to the heart of an individual,wherein said one or more compositions comprises an agent that partiallyor completely downregulates or inactivates p63, p53, and/or p21. Inparticular embodiments, the agent partially or completely downregulatesor inactivates expression of p63, p53, and/or p21. In specificembodiments, the agent comprises p63 shRNA or siRNA, p53 shRNA or siRNA,or p21 shRNA or siRNA, respectively. Any isoform of p63 may be partiallyor completely downregulated or inactivated, including TA, for example.

In embodiments of the disclosure, methods comprise the step of providingto the individual an effective amount of one or more cardiac cellreprogramming factors, which may be a polypeptide, peptide, nucleic acidor mixture thereof. In specific embodiments, the one or more cardiaccell reprogramming factors is Hand2, myocardin, Gata4, Mef2c, Tbx5,Mesoderm posterior protein 1 (Mesp1), miR-133, miR-1, Oct4, Klf4, c-myc,Sox2, Brachyury, Nkx2.5, ETS2, ESRRG, Mrtf-A, MyoD, ZFPM2, miR-590,miR-208, miR-499, or a combination thereof. In certain embodiments, theone or more cardiac cell reprogramming factors is one or both of Hand2and myocardin nucleic acids or polypeptides. In some cases, the one orboth of Hand2 and myocardin nucleic acids or polypeptides are in thesame or different composition as the agent that partially or completelydownregulates or inactivates p63, p53, and/or p21. In specificembodiments, the one or more compositions comprise the nucleic acids ofp63 shRNA and Hand2, the nucleic acids of p63 shRNA and myocardin,and/or the nucleic acids of p63 shRNA, Hand2, and myocardin.

In certain embodiments, the agent that partially or completelydownregulates or inactivates p63, p53, and/or p21 is provided before theone or more cardiac cell reprogramming factors. In particularembodiments of the method, an effective amount of one or more chromatindestabilizing agents are provided to the individual. In particularembodiments, the one or more chromatin destabilizing agents are providedto the individual prior to when the agent that partially or completelydownregulates or inactivates p63, p53, and/or p21 is provided to theindividual. In some embodiments, the one or more chromatin destabilizingagents are provided to the individual prior to when the agent thatpartially or completely downregulates or inactivates p63, p53, and/orp21 is provided to the individual, and wherein the agent that partiallyor completely downregulates or inactivates p63, p53, and/or p21 isprovided to the individual prior to when the one or more cardiac cellreprogramming factors are provided to the individual.

In certain embodiments, the cardiac cells are fibroblasts, endothelialcells, myoblasts, progenitor cells, stem cells, myofibroblasts, or acombination thereof. The cardiac cell may be a dividing cell or anon-dividing cell.

In particular embodiments, the agent that partially or completelydownregulates or inactivates p63, p53, and/or p21 comprises a nucleicacid and said nucleic acid is comprised on one or more vectors. One ormore cardiac cell reprogramming factors may comprise a nucleic acid andthe nucleic acid may be comprised on one or more vectors. In specificembodiments, one or more chromatin destabilizing agents comprise anucleic acid and the nucleic acid is comprised on one or more vectors.In some embodiments, the nucleic acids are comprised on separate vectorsor on the same vector. In certain cases, the vector is a viral vector ora non-viral vector, such as a nanoparticle, plasmid, liposome, or acombination thereof. In a specific embodiment, the viral vector is anadenoviral, lentiviral, retroviral, adeno-associated viral vector, orepisomal (non-integrating) vectors. In particular embodiments, p63shRNA, Hand2, and/or myocardin nucleic acids are comprised on alentiviral vector or are comprised on an adenoviral vector or are amodified mRNA molecule. In any vector encompassed by the disclosure,there may be a cell-specific promoter, such as a fibroblast-specificpromoter.

In specific embodiments, any method encompassed by the disclosurecomprises the step of delivering to the individual an additional cardiactherapy, such as one that comprises drug therapy, surgery, ventricularassist device (VAD) implantation, video assisted thoracotomy (VAT)coronary bypass, percutaneous coronary intervention (PCI), or acombination thereof.

Any of the compositions encompassed by the disclosure may be provided tothe individual in a suitable delivery route, including systemic or localdelivery. In specific embodiments, the delivery is local to the heart,and in specific embodiments, the providing step is further defined asinjecting the compound(s) into the heart.

In one embodiment, there is a composition comprising one or more nucleicacid vectors, the vectors comprising one or more agents that partiallyor completely downregulates or inactivates p63, p53, and/or p21 andcomprising one or more cardiac cell reprogramming factors. In somecases, the vector comprising one or more agents that partially orcompletely downregulates or inactivates p63, p53, and/or p21 is the samevector that comprises one or more cardiac cell reprogramming factors. Incertain embodiments, the vector comprising one or more agents thatpartially or completely downregulates or inactivates p63, p53, and/orp21 is a different vector than the vector that comprises one or morecardiac cell reprogramming factors. In specific embodiments, a vectorfurther comprise one or more chromatin destabilizing agents. The vectorthat comprises one or more agents that partially or completelydownregulates or inactivates p63, p53, and/or p21 may be the same vectorthat comprises one or more chromatin destabilizing agents. The vectorthat comprises one or more agents that partially or completelydownregulates or inactivates p63, p53, and/or p21 and that comprises oneor more cardiac cell reprogramming factors may be the same vector thatcomprises one or more chromatin destabilizing agents. The vector thatcomprises one or more agents that partially or completely downregulatesor inactivates p63, p53, and/or p21 and that comprises one or morecardiac cell reprogramming factors may be a different vector thatcomprises one or more chromatin destabilizing agents. The compositionthat comprises a vector may comprise p63 shRNA and one or both of Hand 2and myocardin nucleic acids. In certain embodiments, the compositionthat comprises a vector comprises p63 shRNA and Hand2 nucleic acids. Insome embodiments, a composition that comprises a vector comprises p63shRNA and myocardin nucleic acids or may comprise p63 shRNA, Hand2, andmyocardin nucleic acids. In specific embodiments, any composition of thedisclosure may comprise one or more anti-fibrotic agents.

In one embodiment there is a kit comprising a composition encompassed bythe disclosure, said composition being housed in a suitable container.

The foregoing has outlined rather broadly the features and technicaladvantages of the present disclosure in order that the detaileddescription of the disclosure that follows may be better understood.Additional features and advantages of the disclosure will be describedhereinafter which form the subject of the claims of the disclosure. Itshould be appreciated by those skilled in the art that the conceptionand specific embodiment disclosed may be readily utilized as a basis formodifying or designing other structures for carrying out the samepurposes of the present disclosure. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the disclosure as set forth in the appendedclaims. The novel features which are believed to be characteristic ofthe disclosure, both as to its organization and method of operation,together with further objects and advantages will be better understoodfrom the following description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an example of experimental design for analyzing fibroblasttransdifferentiation into cardiomyocytes in vitro. GMT refers to thecombination of Gata4, Mef2c, and Tbx5. GFP refer to green fluorescenceprotein. MEF(s) refers to mouse embryonic fibroblast(s). p63 −/−indicates MEFs which have had the p63 gene deleted (Exons 6-8, DNAbinding domain). Reprogramming efficiency was measured with FlowCytometry for cardiac troponin T (cTnT) expression.

FIG. 2 shows fluorescence activated cell sorting (FACS) analysis of apositive control comprising neonatal rat cardiomyoblasts.

FIG. 3 demonstrates a negative control using wild-type cells given novirus.

FIG. 4 shows wild-type cells that are exposed to GFP.

FIG. 5 demonstrates wild-type cells that were given GMT.

FIG. 6 shows a negative control for p63−/− mouse embryonic cells thatwere only stained with the secondary antibody. This was one of thecontrols used to set the gate for FACS analysis.

FIG. 7 demonstrates p63−/− cells that were stained with both the primaryand secondary antibodies.

FIG. 8 demonstrates p63−/− cells that were given GFP and stained withonly the secondary antibody. This was one of the controls used to setthe gate for FACS analysis.

FIG. 9 shows p63−/− cells that were given GFP and stained with both theprimary and secondary antibodies.

FIG. 10 shows FACS results for p63−/− cells given GMT.

FIG. 11 illustrates all results as a function of cTnT expression, whichis a marker of cardiomyocyte lineage.

FIG. 12 illustrates an example of an experimental design for analyzingtransdifferentiation of fibroblasts into cardiomyocytes in vitro.Specifically, the inventors analyzed the role of the two isoforms of thep63 gene. “p63−/−” indicates MEFs that have had the both isoforms of thep63 gene deleted. Reprogramming efficiency was measured with FlowCytometry for cardiac troponin T (cTnT) expression.

FIG. 13 shows FACS data for unstained and stained wild-type mouseembryonic fibroblasts (negative controls).

FIG. 14 shows wild type MEFs treated with lentiviral GFP and lentiviralGMT.

FIG. 15 shows wild-type MEFs that were given certain cardiac cellreprogramming factors, lentiviral Hand2 & Myocardin with or withoutlentiviral miR-590.

FIG. 16 demonstrates FACS analysis for the positive control h9c2neonatal rat cardiomyoblasts.

FIG. 17 shows results for the “p63 −/− MEFs” that concerns theinactivation of both the TA and Delta isoforms of the p63 gene. Thesecells were not treated with reprogramming factors. The left side showsthe p63−/− cells stained with only the secondary antibody, which wasused to set the gate for FACS analysis. The right shows the p63 −/−cells stained with both antibodies.

FIG. 18 demonstrates p63−/− MEFs exposed to GFP. The left shows stainingwith only the secondary antibody, which was used to set the gate forFACS analysis; while the right shows staining with both antibodies.

FIG. 19 shows results of the double KO p63−/− MEFs that were givenlentiviral GMT factors.

FIG. 20 demonstrates double KO p63−/− MEFs exposed to lentiviral Hand2and myocardin, in the presence or absence of miR-590.

FIG. 21 illustrates delta Np63−/− MEFs (knockout of only one isoform)either with no factor, GFP, or GMT.

FIG. 22 shows TAp63−/− MEFs (knockout of only the other isoform) thatwere exposed to either no factor, GFP, or GMT.

FIG. 23 summarizes the study, illustrating the percentage of cellsexpressing cTnT as a marker for cardiomyocyte lineage.

FIG. 24 demonstrates the expression of certain pro-cardiogenic factorsas measured by qPCR of p63 knockout MEFs (H/M=human Hand2/Myocardinlentivirus (SystemsBio)

FIG. 25 illustrates an example of a lentivirus vector with Hand2,Myocardin, p63 and shRNA. An example of such a vector is a genericlentiviral shRNA model from ThermoScientific. RRE: Rev response elementenhances titer by increasing packaging efficiency of full length viralgenomes; CMV promoter: Human CMV promoter drives strong transgeneexpression; Hand2: Transcription factor; IRES: Internal ribosome entrysite.

FIG. 26 shows downregulation of Snail expression in rat cardiacfibroblasts treated with GMT; qPCR analysis of Slug and Snail expressionis shown. After 14 days treatment with human lentiviral GMT or GFPalone, rat cardiac fibroblasts were analyzed for levels of the mRNAs(n=2 biological replicates, each performed in triplicate). Data areshown as fold change relative to day0. RCF: rat cardiac fibroblast.Error bars show SEM.

FIG. 27 illustrates an example protocol of how p63 can be knocked downusing shRNA in mouse embryonic fibroblasts (MEFs) in vitro.

FIG. 28 demonstrates p63 shRNA knockdown efficiency in MEFs.

FIG. 29 demonstrates that p63 knockdown MEFs, i.e. those treated withshRNA, also express pro-cardiogenic factors Gata4, Mef2c, Tbx5, Hand2and Myocardin (tested at three weeks).

FIG. 30 demonstrates p63 knockdown efficiency in adult humankeratinocytes. P63 knockdown also results in upregulation of cTnt, whichis further enhanced with Hand2, Myocardin.

FIG. 31 shows that adult human keratinocytes treated with p63 shRNAlentivirus express pro-cardiogenic markers as well as cardiac structuralmarkers Myosin Heavy Chain 6 (MYH6) and Myosin Heavy Chain 7 (MYH7).

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the use of the word “a” or “an” when used in conjunctionwith the term “comprising” in the claims and/or the specification maymean “one,” but it is also consistent with the meaning of “one or more,”“at least one,” and “one or more than one.” Some embodiments of thedisclosure may consist of or consist essentially of one or moreelements, method steps, and/or methods of the disclosure. It iscontemplated that any method or composition described herein can beimplemented with respect to any other method or composition describedherein.

The term “cardiac medical condition” as used herein refers to anymedical condition that affects heart tissue, including that affectsheart function.

The term “chromatin destabilizing agent” as used herein refers to one ormore compounds that facilitate access of one or more factors tocondensed genomic DNA.

The term “cardiac cell reprogramming factor” as used herein refers toone or more compositions that enhance or facilitate thetransdifferentiation of a differentiated cell in the heart to acardiomyocyte.

Embodiments of the disclosure include methods and compositions for thetherapy or prevention of any cardiac medical condition in which it wouldbe therapeutic to increase the number of cardiomyocytes in the heart. Inspecific embodiments, in vivo cells in the heart are reprogrammed tobecome cardiomyocytes. In particular embodiments, this is achieved atleast in part by inactivating p63, p53, and/or p21, including at thenucleic acid level. In particular embodiments, nucleic acids and/orpeptides and/or polypeptides are delivered directly to the heart toallow for reprogramming of non-cardiomyocyte cells in the heart tobecome cardiomyocytes.

I. Embodiments of Methods of Treatment

Embodiments of the present disclosure are directed to methods and/orcompositions related to therapy and/or prevention of one or morecardiac-related medical conditions. Embodiments of the presentdisclosure concern regeneration of tissue, including muscle tissue, suchas myocardial tissue, through the reprogramming of existing cells in theheart that are not cardiomyocytes. Certain embodiments relate toreversal of a cardiac medical condition (or improvement of at least onesymptom thereof), including at least cardiac disease, cardiomyopathy,cardiotoxicity, congestive heart failure, ischemic heart disease,myocardial infarction, coronary artery disease, cor pulmonale,inflammatory heart disease; inflammatory cardiomegaly; myocarditis;congenital heart disease; rheumatic heart disease, cardiac systolicdysfunction, cardiac diastolic dysfunction, angina, dilatedcardiomyopathy, idiopathic cardiomyopathy, or other conditions resultingin cardiac fibrosis, for example.

In particular aspects of the disclosure, cardiomyopathy is the cardiacmedical condition to be treated. The cardiac medical condition(including, for example, cardiomyopathy) may be caused by one or more ofa variety of characteristics, including, for example, long-term highblood pressure; heart valve problems; heart tissue damage (such as fromone or more previous heart attack(s) or chronic or acute and/orrecurrent episodes or sequelae of ischemic heart disease); chronic rapidheart rate; metabolic disorders, such as thyroid disease or diabetes;nutritional deficiencies of essential vitamins or minerals, such asthiamin (vitamin B-1), selenium, calcium and/or magnesium; pregnancy;alcohol abuse; drug abuse, including of narcotics or prescription drugs,such as cocaine or antidepressant medications, such as tricyclicantidepressants; use of some chemotherapy drugs to treat cancer(including Adriamycin); certain viral infections; hemochromatosis and/oran unknown cause or undetected cause, i.e. idiopathic cardiomyopathy.

In some cases, methods and compositions of the present disclosure areemployed for treatment or prevention of one or more cardiac medicalconditions or delay of onset of one or more cardiac medical conditionsor reduction of extent of one or more symptoms of one or more cardiacmedical conditions. In particular cases, such prevention, delay oronset, or reduction of extent of one or more symptoms, occurs in anindividual that is at risk for a cardiac medical condition. Exemplaryrisk factors include one or more of the following: age, gender (male,although it occurs in females), high blood pressure, high serumcholesterol levels, tobacco smoking, excessive alcohol consumption,sugar consumption, family or personal history, obesity, lack of physicalactivity, psychosocial factors, diabetes mellitus, overweight, geneticpredisposition, and/or exposure to air pollution.

Particular aspects of the disclosure concern delivery of at least onepolynucleotide (including a gene), small molecule, peptide, polypeptide,shRNA polynucleotide, siRNApolynucleotide, and so forth to cardiactissue for trans-differentiation of certain cells in the tissue. Inspecific embodiments, a nucleic acid is the active agent, whereas insome embodiments a polypeptide produced from the nucleic acid is theactive agent. The tissue may be of any kind, but in specific cases it iscardiac muscle and/or scar tissue. In particular embodiments, methodsand compositions of the disclosure allow for differentiation of adultresident cardiac progenitor cells and/or transdifferentiation ofnon-cardiomyocyte differentiated cells, such as fibroblast cells, intocardiac muscle cells.

Embodiments of the disclosure include delivery of one or morepolynucleotides (which may also be referred to as nucleic acids) orpolypeptides produced therefrom that stimulate transdifferentiation ordirect reprogramming of cells (such as muscle cells, includingcardiomyocytes) and/or tissue (including cardiac tissue). Particularaspects for such embodiments result in reversal of one or more cardiacmedical conditions. Certain aspects for such embodiments result inimprovement of at least one symptom of a cardiac medical condition. Inexemplary embodiments, the cardiac medical condition is heart failure.The heart failure may be the result of one or more causes, includingcoronary artery disease and heart attack, high blood pressure, faultyheart valves, cardiomyopathy (such as caused by disease, infection,alcohol abuse and the toxic effect of drugs, such as cocaine or somedrugs used for chemotherapy), idiopathic cardiomyopathy and/or geneticfactors.

Particular but exemplary indications of embodiments of the disclosureinclude at least applications for 1) heart failure, including congestiveheart failure; 2) prevention of ventricular remodeling; and/or 3)cardiomyopathy. Other indications may also include coronary arterydisease, ischemic heart disease, valvular heart disease, etc. Inspecific embodiments, methods and compositions of the disclosure providecardiomyocyte regeneration that is sufficient to reverse establishedcardiomyopathy, congestive heart failure, and prevention of ventricularremodeling.

In cases where the individual has cardiomyopathy, the cardiomyopathy maybe ischemic or non-ischemic cardiomyopathy. The cardiomyopathy may becaused by long-term high blood pressure, heart valve problems, hearttissue damage from a previous heart attack, chronic rapid heart rate,metabolic disorders, nutritional deficiencies, pregnancy, alcohol abuse,drug abuse, chemotherapy drugs, viral infection, hemochromatosis,genetic condition, elevated cholesterol levels, or a combinationthereof. Cardiomyopathy may also have no identified cause, i.e.idiopathic cardiomyopathy.

In certain embodiments, there is a method of regenerating cells at adesired location in an individual, comprising the steps of delivering tothe location an effective amount of at least one molecule for p63, p53,and/or p21 inactivation. The inactivation of p63, p53, and/or p21 may bepartial or complete. The at least one molecule for p63, p53, and/or p21inactivation may be of any kind, such as p63 shRNA or siRNA, p53 shRNAor siRNA, and/or p21 shRNA or siRNA, respectively, with or without oneor more cardiac cell reprogramming factor (such as Hand2 and/ormyocardin, for example). In specific embodiments, the molecules aredelivered in nucleic acid form, although in specific embodiments one ormore of the compositions of the disclosure that are not directed to p63,p53, and/or p21 interference are polypeptides. In particularembodiments, the delivery location of the composition(s) is at a regionof the heart. A delivering step may comprise injection directly into theheart, including directly into the area of damaged tissue; intravenousperfusion; intra-coronary artery myocardium perfusion; intra-arteryorgan perfusion by catheter; or coronary sinus perfusion catheter, forexample.

Embodiments of the disclosure include methods and/or compositions forregeneration of cardiac muscle and reversal of myocardial ischemicinjury, for example. In particular embodiments, there are methods forreprogramming of cardiac scar cells (fibroblasts) into adult cardiacmuscle cells in mammalian hearts in an individual that has had a cardiacmedical condition, such as acute or chronic ischemic injury, forexample. In certain embodiments, such methods are achieved withcompositions comprising at least p63 shRNA or siRNA, p53 shRNA or siRNA,and/or p21 shRNA or siRNA (as an example of RNA interference) and, insome cases, one or more cardiac cell reprogramming factors, such asHand2 and/or myocardin, for example.

Although in particular embodiments an individual is treated in an invivo or in situ manner, in alternative embodiments the individual istreated with compositions encompassed by the disclosure in an ex vivomanner. In such embodiments, cells that are to be subjected to nucleicacid composition(s) of the disclosure are either obtained from theindividual or are obtained from another individual. Such cells aresubjected in vitro to the nucleic acid compositions such that they areuptaken by the cells, and the cells are then delivered to the individualto be treated.

In particular aspects, an individual is provided with an additionalcardiac medical condition therapy.

II. p63, p53, and/or p21 Inactivation

In embodiments of the disclosure, the inactivation of p63, p53, and/orp21 is utilized for individuals with a cardiac medical condition tofacilitate the reprogramming of endogenous cells in the heart to becomecardiomyocytes, thereby regenerating cardiac tissue. The inactivation ofp63, p53, and/or p21 may occur by any means, including at least by RNAinterference in some manner, and in some cases with shRNA or siRNA. Assuch, the p63, p53, and/or p21 becomes partially or completelydownregulated or inactivated by partial or complete knock down.

As used herein, the term “knock-down” or “knock-down technology” refersto a technique of gene silencing in which the expression of p63, p53,and/or p21 is reduced as compared to the gene expression prior to theintroduction of the siRNA or shRNA, which can lead to the inhibition ofproduction of the target gene product. The term “reduced” is used hereinto indicate that the target gene expression is lowered by 0.1-100%. Forexample, the expression may be reduced by 0.5, 1, 5, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or even 99%. Theexpression may be reduced by any amount (%) within those intervals, suchas for example, 2-4, 11-14, 16-19, 21-24, 26-29, 31-34, 36-39, 41-44,46-49, 51-54, 56-59, 61-64, 66-69, 71-74, 76-79, 81-84, 86-89, 91-94,96, 97, 98 or 99. Knock-down of gene expression can be directed by theuse of siRNAs or shRNAs.

As used herein, the terms “small interfering” or “short interfering RNA”or “siRNA” refer to an RNA duplex of nucleotides that is targeted to thep63, p53, and/or p21 gene, respectively, and is capable of inhibitingthe expression of the p63 gene. The RNA duplex comprises twocomplementary single-stranded RNAs of 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or 25 nucleotides that form 15, 16, 17, 18, 19, 20, 21, 22, 23,24, or 25 base pairs and possess 3′ overhangs of two nucleotides. TheRNA duplex is formed by the complementary pairing between two regions ofa RNA molecule. siRNA is “targeted” to p63, p53, and/or p21 in that thenucleotide sequence of the duplex portion of the siRNA is complementaryto a nucleotide sequence of p63, p53, and/or p21. In some embodiments,the length of the duplex of siRNAs is less than 30 nucleotides. Theduplex can be 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16,15, 14, 13, 12, 11 or 10 nucleotides in length. The length of the duplexcan be 17-25 nucleotides in length. The duplex RNA can be expressed in acell from a single construct.

As used herein, the term “shRNA” (small hairpin RNA) refers to an RNAduplex wherein a portion of the siRNA is part of a hairpin structure(shRNA). In addition to the duplex portion, the hairpin structure maycontain a loop portion positioned between the two sequences that formthe duplex. The loop can vary in length. In some embodiments the loop is5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleotides in length. The hairpinstructure can also contain 3′ or 5′ overhang portions. In some aspects,the overhang is a 3′ or a 5′ overhang 0, 1, 2, 3, 4 or 5 nucleotides inlength. In one aspect of this disclosure, a nucleotide sequence in thevector serves as a template for the expression of a small hairpin RNA,comprising a sense region, a loop region and an antisense region thattargets p63, p53, and/or p21. Following expression, the sense andantisense regions form a duplex. It is this duplex, forming the shRNA,which hybridizes to, for example, the p63 mRNA and reduces expression ofp63, p53, and/or p21. See Moore et al., Methods Mol Biol. 2010; 629:141-158, for disclosure of shRNA design.

According to the disclosure, an siRNA or shRNA corresponding to a regionof p63, p53, and/or p21 is expressed in the cell. The RNA duplex issubstantially identical (typically at least about 80% identical, andmore typically at least about 90% identical) in sequence to a region ofp63, p53, and/or p21. siRNA duplexes are described and well known in theart. See, for example, U.S. Pat. No. 7,410,944.

In one embodiment, the shRNA is a “hairpin” or stem-loop RNA molecule,comprising a sense region, a loop region and an antisense regioncomplementary to the sense region. In other embodiments the shRNAcomprises two distinct RNA molecules that are non-covalently associatedto form a duplex. See, for example, U.S. Pat. No. 7,195,916.

When appropriately targeted via its nucleotide sequence to p63, p53,and/or p21 mRNA in cells, an siRNA or shRNA can specifically suppressgene expression through a process known as RNA interference (RNAi).siRNAs or shRNAs can reduce the cellular level of specific p63, p53,and/or p21 mRNAs, respectively, and decrease the level of proteins codedby such mRNAs. siRNAs and shRNAs utilize sequence complementarity totarget an mRNA for destruction, and are sequence-specific. Thus, theycan be highly target-specific, and in mammals have been shown to targetmRNAs encoded by different alleles of the same gene.

It should further be noted that full complementarity between the targetsequence and the antisense siRNA or shRNA is not required. That is, theresultant antisense siRNA or shRNA is sufficiently complementary withthe target sequence. The sense strand is substantially complementarywith the antisense strand to anneal (hybridize) to the antisense strandunder biological conditions.

In particular, the complementary polynucleotide sequence of shRNA can bedesigned to specifically hybridize to a particular region of p63, p53,and/or p21 mRNA to interfere with replication, transcription, ortranslation. The term “hybridize” or variations thereof, refers to asufficient degree of complementarity or pairing between an antisensenucleotide sequence and p63, p53, and/or p21 DNA or mRNA such thatstable and specific binding occurs there between. In particular, 100%complementarity or pairing is desirable but not required. Specifichybridization occurs when sufficient hybridization occurs between theantisense nucleotide sequence and p63 nucleic acids in the substantialabsence of non-specific binding of the antisense nucleotide sequence tonon-target sequences under predetermined conditions, e.g., for purposesof in vivo treatment, preferably under physiological conditions.Preferably, specific hybridization results in the interference withnormal expression of the p63, p53, and/or p21 gene product encoded bythe target p63, p53, and/or p21 DNA or mRNA.

For example, a p63, p53, and/or p21 antisense nucleotide sequence can bedesigned to specifically hybridize to the replication or transcriptionregulatory regions of p63, or the translation regulatory regions such astranslation initiation region and exon/intron junctions, or the codingregions of a p63, p53, and/or p21 mRNA.

p63, p53, and/or p21 siRNA or shRNA: Synthesis

As is generally known in the art, commonly used oligonucleotides areoligomers or polymers of ribonucleic acid or deoxyribonucleic acidhaving a combination of naturally-occurring purine and pyrimidine bases,sugars and covalent linkages between nucleosides including a phosphategroup in a phosphodiester linkage. However, it is noted that the term“oligonucleotides” also encompasses various non-naturally occurringmimetics and derivatives, i.e., modified forms, of naturally-occurringoligonucleotides as described below.

p63, p53, and/or p21 siRNA or shRNA molecules of the invention can beprepared by any method known in the art for the synthesis of DNA and RNAmolecules. These include techniques for chemically synthesizingoligodeoxy-ribonucleotides and oligo-ribonucleotides well known in theart such as for example solid phase phosphoramidite chemical synthesis.Alternatively, RNA molecules can be generated by in vitro and in vivotranscription of DNA sequences encoding the antisense RNA molecule. SuchDNA sequences may be incorporated into a wide variety of vectors thatincorporate suitable RNA polymerase promoters such as the T7 or SP6polymerase promoters. Alternatively, antisense cDNA constructs thatsynthesize antisense RNA constitutively or inducibly, depending on thepromoter used, can be introduced stably into cell lines.

p63, p53, and/or p21 siRNA or shRNA molecules can be chemicallysynthesized using appropriately protected ribonucleosidephosphoramidites and a conventional DNA/RNA synthesizer. Custom siRNAsynthesis services are available from commercial vendors such as Ambion(Austin, Tex., USA) and Dharmacon Research (Lafayette, Colo., USA). See,for example, U.S. Pat. No. 7,410,944.

Various well-known modifications to the DNA molecules can be introducedas a means of increasing intracellular stability and half-life. Possiblemodifications include, but are not limited to, the addition of flankingsequences of ribo- or deoxy-nucleotides to the 5′ and/or 3′ ends of themolecule or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the oligodeoxyribonucleotide backbone.An antisense nucleic acid of the disclosure can be constructed usingchemical synthesis or enzymatic ligation reactions using proceduresknown in the art. An antisense oligonucleotide can be chemicallysynthesized using naturally-occurring nucleotides or variously modifiednucleotides designed to increase the biological stability of themolecules or to increase the physical stability of the duplex formedbetween the antisense and sense nucleic acids (e.g., phosphorothioatederivatives and acridine substituted nucleotides can be used).

The p63, p53, and/or p21 siRNA or shRNA molecules of the disclosure canbe various modified equivalents of the structures of any p63, p53,and/or p21 siRNA or shRNA. A “modified equivalent” means a modified formof a particular siRNA or shRNA molecule having the sametarget-specificity (i.e., recognizing the same mRNA molecules thatcomplement the unmodified particular siRNA molecule). Thus, a modifiedequivalent of an unmodified siRNA or shRNA molecule can have modifiedribonucleotides, that is, ribonucleotides that contain a modification inthe chemical structure of an unmodified nucleotide base, sugar and/orphosphate (or phosphodiester linkage). See, for example, U.S. Pat. No.7,410,944.

Preferably, modified p63, p53, and/or p21 siRNA or shRNA moleculescontain modified backbones or non-natural internucleoside linkages,e.g., modified phosphorous-containing backbones and non-phosphorousbackbones such as morpholino backbones; siloxane, sulfide, sulfoxide,sulfone, sulfonate, sulfonamide, and sulfamate backbones; formacetyl andthioformacetyl backbones; alkene-containing backbones; methyleneiminoand methylenehydrazino backbones; amide backbones, and the like. See,for example, U.S. Pat. No. 7,410,944.

Examples of modified phosphorous-containing backbones include, but arenot limited to phosphorothioates, phosphorodithioates, chiralphosphorothioates, phosphotriesters, aminoalkylphosphotriesters, alkylphosphonates, thionoalkylphosphonates, phosphinates, phosphoramidates,thionophosphoramidates, thionoalkylphosphotriesters, andboranophosphates and various salt forms thereof. See, for example, U.S.Pat. No. 7,410,944.

Examples of the non-phosphorous containing backbones described above areknown in the art, e.g., U.S. Pat. No. 5,677,439, each of which is hereinincorporated by reference. See, for example, U.S. Pat. No. 7,410,944.

Modified forms of p63, p53, and/or p21 siRNA or shRNA compounds can alsocontain modified nucleosides (nucleoside analogs), i.e., modified purineor pyrimidine bases, e.g., 5-substituted pyrimidines, 6-azapyrimidines,pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxybenzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl,5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g.,ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidinesor 6-alkylpyrimidines (e.g. 6-methyluridine), 2-thiouridine,4-thiouridine, 5-(carboxyhydroxy methyl)uridine,5′-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluridine, 5-methoxyaminomethyl-2-thiouridine,5-methylaminomethyluridine, 5-methylcarbonylmethyl uridine,5-methyloxyuridine, 5-methyl-2-thiouridine, 4-acetylcytidine,3-methylcytidine, propyne, quesosine, wybutosine, wybutoxosine,beta-D-galactosylqueosine, N-2, N-6 and O-substituted purines, inosine,1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine,2-methyladenosine, 2-methylguanosine, N6-methyladenosine,7-methylguanosine, 2-methylthio-N-6-isopentenyl adenosine,beta-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine,threonine derivatives, and the like. See, for example, U.S. Pat. No.7,410,944. In addition, modified siRNA compounds can also havesubstituted or modified sugar moieties, e.g., 2′-O-methoxyethyl sugarmoieties. See, for example, U.S. Pat. No. 7,410,944. In specificembodiments, the 3′ overhangs of the siRNAs or shRNAs of the presentdisclosure are modified to provide resistance to cellular nucleases. Inone embodiment the 3′ overhangs comprise 2′-deoxyribonucleotides.

Additionally, to assist in the design of p63, p53, and/or p21 siRNAs orshRNAs for the efficient RNAi-mediated silencing of any target gene,several siRNA or shRNA supply companies maintain web-based design toolsthat utilize these general guidelines for selecting siRNAs or shRNAswhen presented with the mRNA or coding DNA sequence of the target gene.Examples of such tools can be found at the web sites of Dharmacon, Inc.(Lafayette, Colo.), Ambion, Inc. (Austin, Tex.). As an example,selecting siRNAs involves choosing a site/sequence unique to the targetgene (i.e., sequences that share no significant homology with genesother than the one being targeted), so that other genes are notinadvertently targeted by the same siRNA designed for this particulartarget sequence.

Another criterion to be considered is whether or not the target sequenceincludes a known polymorphic site. If so, siRNAs or shRNAs designed totarget one particular allele may not effectively target another allele,since single base mismatches between the target sequence and itscomplementary strand in a given siRNA or shRNA can greatly reduce theeffectiveness of RNAi induced by that respective siRNA or shRNA. Giventhat target sequence and such design tools and design criteria, anordinarily skilled artisan apprised of the present disclosure should beable to design and synthesized additional siRNA or shRNA compoundsuseful in reducing the mRNA level of p63.

p63, p53, and/or p21 siRNA or shRNA: Administration

The present invention provides a composition of a polymer or excipientand one or more vectors encoding one or more p63, p53, and/or p21 siRNAor shRNA molecules. The vector can be formulated into a pharmaceuticalcomposition with suitable carriers and administered into a mammal usingany suitable route of administration, including injection into theheart, for example.

Because of this precision, side effects typically associated withtraditional drugs can be reduced or eliminated. In addition, siRNAs orshRNAs are relatively stable, and like antisense, they can also bemodified to achieve improved pharmaceutical characteristics, such asincreased stability, deliverability, and ease of manufacture. Moreover,because siRNA or shRNA molecules take advantage of a natural cellularpathway, i.e., RNA interference, they are highly efficient in destroyingtargeted mRNA molecules. As a result, it is relatively easy to achieve atherapeutically effective concentration of an siRNA or shRNA compound ina subject. See, for example, U.S. Pat. No. 7,410,944.

siRNA or shRNA compounds may be administered to mammals by variousmethods through different routes. They can also be delivered directly toa particular organ or tissue by any suitable localized administrationmethods such as direct injection into a target tissue. Alternatively,they may be delivered encapsulated in liposomes, by iontophoresis, or byincorporation into other vehicles such as hydrogels, cyclodextrins,biodegradable nanocapsules, and bioadhesive microspheres.

In vivo inhibition of specific gene expression by RNAi injectedintravenously has been achieved in various organisms including mammalsOne route of administration of shRNA molecules of the disclosureincludes direct injection of the vector at a desired tissue site, suchas for example, into diseased or non-diseased cardiac tissue, intoischemic heart tissue.

In one aspect of the invention, one or more vectors comprising one ormore of shRNA of the invention can be re-administered an unlimitednumber of times after a first administration at any time interval orintervals after the first administration.

p63, p53, and/or p21 siRNA or shRNA: Pharmaceutical Compositions

The p63, p53, and/or p21 siRNA or shRNA encoding nucleic acids of thepresent invention can be formulated in pharmaceutical compositions,which are prepared according to conventional pharmaceutical compoundingtechniques. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed.(1990, Mack Publishing Co., Easton, Pa.). The pharmaceuticalcompositions of the invention comprise a therapeutically effectiveamount of the vector encoding shRNA. These compositions can comprise, inaddition to the vector, a pharmaceutically acceptable excipient,carrier, buffer, stabilizer or other materials well known in the art.Such materials should be non-toxic and should not interfere with theefficacy of the active ingredient. The carrier can take a wide varietyof forms depending on the form of preparation desired foradministration, e.g., intravenous, oral, intramuscular, subcutaneous,intrathecal, epineural or parenteral.

When the vectors of the disclosure are prepared for administration, theymay be combined with a pharmaceutically acceptable carrier, diluent orexcipient to form a pharmaceutical formulation, or unit dosage form. Thetotal active ingredients in such formulations include from 0.1 to 99.9%by weight of the formulation

In another aspect of the invention, the vectors of the disclosure can besuitably formulated and introduced into the environment of the cell byany means that allows for a sufficient portion of the sample to enterthe cell to induce gene silencing, if it is to occur. Many formulationsfor vectors are known in the art and can be used so long as the vectorsgain entry to the target cells so that it can act.

For example, the vectors can be formulated in buffer solutions such asphosphate buffered saline solutions comprising liposomes, micellarstructures, and capsids. The pharmaceutical formulations of the vectorsof the invention can also take the form of an aqueous or anhydroussolution or dispersion, or alternatively the form of an emulsion orsuspension. The pharmaceutical formulations of the vectors of thepresent invention may include, as optional ingredients, solubilizing oremulsifying agents, and salts of the type that are well-known in theart. Specific non-limiting examples of the carriers and/or diluents thatare useful in the pharmaceutical formulations of the present inventioninclude water and physiologically acceptable saline solutions. Otherpharmaceutically acceptable carriers for preparing a composition foradministration to an individual include, for example, solvents orvehicles such as glycols, glycerol, or injectable organic esters. Apharmaceutically acceptable carrier can contain physiologicallyacceptable compounds that act, for example, to stabilize or to increasethe absorption of the shRNA encoding vector. Other physiologicallyacceptable carriers include, for example, carbohydrates, such asglucose, sucrose or dextrans, antioxidants, such as ascorbic acid orglutathione, chelating agents, low molecular weight proteins or otherstabilizers or excipients, saline, dextrose solutions, fructosesolutions, ethanol, or oils of animal, vegetative or synthetic origin.The carrier can also contain other ingredients, for example,preservatives.

It will be recognized that the choice of a pharmaceutically acceptablecarrier, including a physiologically acceptable compound, depends, forexample, on the route of administration of the composition. Thecomposition containing the vectors can also contain a second reagentsuch as a diagnostic reagent, nutritional substance, toxin, oradditional therapeutic agent. Many agents useful in the treatment ofcardiac disease are known in the art and are envisioned for use inconjunction with the vectors of this invention.

Formulations of vectors with cationic lipids can be used to facilitatetransfection of the vectors into cells. For example, cationic lipids,such as lipofectin, cationic glycerol derivatives, and polycationicmolecules, such as polylysine, can be used. Suitable lipids include, forexample, Oligofectamine and Lipofectamine (Life Technologies) which canbe used according to the manufacturer's instructions.

Suitable amounts of vector must be introduced and these amounts can beempirically determined using standard methods. Typically, effectiveconcentrations of individual vector species in the environment of a cellwill be about 50 nanomolar or less 10 nanomolar or less, or compositionsin which concentrations of about 1 nanomolar or less can be used. Inother aspects, the methods utilize a concentration of about 200picomolar or less and even a concentration of about 50 picomolar or lesscan be used in many circumstances. One of skill in the art can determinethe effective concentration for any particular mammalian subject usingstandard methods.

The p63, p53, and/or p21 siRNA or shRNA is administered in atherapeutically effective amount. The actual amount administered, andthe rate and time-course of administration, will depend on the natureand severity of the condition, disease or disorder being treated.Prescription of treatment, for example, decisions on dosage, timing,etc., is within the responsibility of general practitioners orspecialists, and typically takes account of the disorder, condition ordisease to be treated, the condition of the individual mammaliansubject, the site of delivery, the method of administration and otherfactors known to practitioners. Examples of techniques and protocols canbe found in Remington's Pharmaceutical Sciences 18th Ed. (1990, MackPublishing Co., Easton, Pa.).

Alternatively, targeting therapies can be used to deliver the shRNAencoding vectors more specifically to certain types of cell, by the useof targeting systems such as antibodies or cell specific ligands.Targeting can be desirable for a variety of reasons, e.g., if the agentis unacceptably toxic, or if it would otherwise require too high adosage, or if it would not otherwise be able to enter the target cells.

p63, p53, and/or p21 siRNA or shRNA: Gene Therapy

p63, p53, and/or p21 siRNA or shRNA can also be delivered into mammaliancells, particularly human cells, by a gene therapy approach, using a DNAvector, for example. In specific embodiments a vector from which siRNAcompounds in, e.g., small hairpin form (shRNA), can be transcribeddirectly is utilized. Studies have demonstrated that whiledouble-stranded siRNAs are very effective at mediating RNAi, short,single-stranded, hairpin-shaped RNAs can also mediate RNAi, presumablybecause they fold into intramolecular duplexes that are processed intodouble-stranded siRNAs by cellular enzymes. This discovery hassignificant and far-reaching implications, since the production of suchshRNAs can be readily achieved in vivo by transfecting cells or tissueswith DNA vectors bearing short inverted repeats separated by a smallnumber of (e.g., 3, 4, 5, 6, 7, 8, 9) nucleotides that direct thetranscription of such small hairpin RNAs. Additionally, if mechanismsare included to direct the integration of the vector or a vector segmentinto the host-cell genome, or to ensure the stability of thetranscription vector, the RNAi caused by the encoded shRNAs, can be madestable and heritable. Not only have such techniques been used to “knockdown” the expression of specific genes in mammalian cells, but they havenow been successfully employed to knock down the expression ofexogenously expressed transgenes, as well as endogenous genes in thebrain and liver of living mice.

Gene therapy is carried out according to generally accepted methods asare known in the art. See, for example, U.S. Pat. Nos. 5,837,492 and5,800,998 and references cited therein. Vectors in the context of genetherapy are meant to include those polynucleotide sequences containingsequences sufficient to express a polynucleotide encoded therein. If thepolynucleotide encodes an shRNA, expression will produce the antisensepolynucleotide sequence. Thus, in this context, expression does notrequire that a protein product be synthesized. In addition to the shRNAencoded in the vector, the vector also contains a promoter functional ineukaryotic cells. The shRNA sequence is under control of this promoter.Suitable eukaryotic promoters include those described elsewhere hereinand as are known in the art. The expression vector may also includesequences, such as selectable markers, reporter genes and otherregulatory sequences conventionally used.

Accordingly, the amount of siRNA generated in situ is regulated bycontrolling such factors as the nature of the promoter used to directtranscription of the nucleic acid sequence, (i.e., whether the promoteris constitutive or regulatable, strong or weak) and the number of copiesof the nucleic acid sequence encoding a siRNA sequence that are in thecell.

III. Embodiments of Cardiac Cell Reprogramming Factors and ChromatinDestabilizing Agents

Certain embodiments of the present disclosure concern nucleic acids, andsome embodiments concern polypeptides or peptides. In certain aspects,nucleic acids include a one or more agents that partially or completelydownregulates or inactivates p63, p53, and/or p21, such as bydownregulating or inactivating their expression. In specificembodiments, the agent comprises RNA interference nucleic acid (such asshRNA or siRNA). In particular embodiments, the agent(s) may or may notbe utilized with one or more cardiac cell reprogramming factors and mayor may not be used with one or more chromatin destabilizing agents.

Cardiac Cell Reprogramming Factors

In specific embodiments, one or more cardiac cell reprogramming factorsare employed in methods of the disclosure, and the factors may or maynot be provided at the same time as the agent that partially orcompletely downregulates or inactivates p63, p53, and/or p21. Inspecific embodiments, the factors are provided after the individual hasreceived the p63, p53, and/or p21-inactivating agent(s), although insome cases they are provided before or at the same time as the agent(s).

The cardiac cell reprogramming factor may or may not be a transcriptionfactor. Although one could use standard methods to test whether or not acertain compound would be effective as a cardiac cell reprogrammingfactor, in specific embodiments the factor is Hand2, myocardin, Gata4,Mef2c, Tbx5, Mesoderm posterior protein 1 (Mesp1), miR-133, miR-1, Oct4,Klf4, c-myc, Sox2, Brachyury, Nkx2.5, ETS2, ESRRG, Mrtf-A, MyoD, ZFPM2,or a combination thereof. One could test whether or not a compound actedas a cardiac cell reprogramming factor by administering it tofibroblasts (e.g., using lentivirus) and perform FACS for cTnT asillustrated elsewhere herein. The factors may be employed as nucleicacids, polypeptides, peptides of specific domains of the factor, or acombination thereof. In specific embodiments, Hand2 and/or myocardin areemployed, including as nucleic acids. In particular aspects at least forHand2 and/or myocardin, the nucleic acid encodes for or comprises atranscribed nucleic acid. In other aspects, a Hand2 and/or myocardinnucleic acid comprises a nucleic acid segment of Hand2 and/or myocardin,respectively, or a biologically functional equivalent thereof. Inparticular aspects, a Hand2 and/or myocardin nucleic acid encodes aprotein, polypeptide, or peptide. An exemplary human Hand2 nucleic acidis at the GenBank® database of National Center for BiotechnologyInformation, Accession No. NM_021973, which is incorporated by referenceherein in its entirety. An exemplary human myocardin nucleic acid is atGenBank® Accession Number AY764180, which is incorporated by referenceherein in its entirety. An exemplary human p63 nucleic acid sequence isat GenBank Accession Number NM_003722, and the skilled artisanrecognizes that interference of p63 nucleic acid would comprise sequencethat is complementary to at least part of the p63, p53, and/or p21 mRNA.In specific embodiments, the p63, p53, and/or p21 shRNA is capable ofDNA integration and comprises two complementary 19-22 bp RNA sequenceslinked by a short loop of 4-11 nt, wherein the region of complementaritymay be to any region of the p63 nucleic acid sequence, including codingor non-coding sequence.

In specific embodiments, a functional fragment of the cardiac cellreprogramming factor nucleic acid or polypeptide is utilized instead ofthe entire factor nucleic acid or polypeptide. A functional fragment ofeither Hand2 or myocardin (as an example) is one that is sufficient toallow reprogramming of cells upon exposure to the fragment, either alonewith p63, p53, and/or p21 shRNA or in conjunction with myocardin andp63, p53, and/or p21 shRNA. In specific embodiments, the functionalfragment of Hand2 nucleic acid encodes at least 200, 180, 175, 160, 150,140, 125, 110, 100, 90, 80, 75, 70, 60, 55, 50, 40, 30, 25, or 19 aminoacids of the Hand2 polypeptide. In specific embodiments, the functionalfragment of myocardin nucleic acid encodes at least 900, 800, 700, 600,500, 400, 300, 200, 100, or 50 amino acids of the myocardin polypeptide.

Chromatin Destabilizing Agents

In particular embodiments, one or more chromatin destabilizing agentsare utilized with one or more p63, p53, and/or p21 inactivating agents.The one or more chromatin destabilizing agents may be provided to anindividual at the same time as the one or more p63, p53, and/or p21inactivating agents, although in specific embodiments the one or morechromatin destabilizing agents are utilized before or after one or morep63, p53, and/or p21 inactivating agents.

Although one could use standard methods to test whether or not a certaincompound would be effective as a chromatin destabilizing agent, inspecific embodiments the chromatin destabilizing agent is Oct4, DZNep,Sall4, SOX2, KLF4, MYC, SB431542, PD0325901, Parnate. CHIR99021,A-83-01. NaB, PS48, Forskolin (FSK), 2-methyl-5-hydroxytryptamine(2-Me-5HT), D4476, VPA, CHIR99021 (CHIR), 616452, Tranylcypromine,Prostaglandin E2, Rolipram, 3-deazaneplanocin A (DZNep), 5-Azacytidine,sodium butyrate, RG108 or a combination thereof.

The term “nucleic acid” is well known in the art. A “nucleic acid” asused herein will generally refer to a molecule (i.e., a strand) of DNA,RNA or a derivative or analog thereof, comprising a nucleobase. Anucleobase includes, for example, a naturally occurring purine orpyrimidine base found in DNA (e.g., an adenine “A,” a guanine “G,” athymine “T” or a cytosine “C”) or RNA (e.g., an A, a G, an uracil “U” ora C). The term “nucleic acid” encompass the terms “oligonucleotide” and“polynucleotide,” each as a subgenus of the term “nucleic acid.” Theterm “oligonucleotide” refers to a molecule of between about 3 and about100 nucleobases in length. The term “polynucleotide” refers to at leastone molecule of greater than about 100 nucleobases in length, in atleast some cases.

These definitions generally refer to a single-stranded molecule, but inspecific embodiments will also encompass an additional strand that ispartially, substantially or fully complementary to the single-strandedmolecule. Thus, a nucleic acid may encompass a double-stranded moleculeor a triple-stranded molecule that comprises one or more complementarystrand(s) or “complement(s)” of a particular sequence comprising amolecule. As used herein, a single stranded nucleic acid may be denotedby the prefix “ss,” a double stranded nucleic acid by the prefix “ds,”and a triple stranded nucleic acid by the prefix “ts.”

A. Nucleobases

As used herein a “nucleobase” refers to a heterocyclic base, such as forexample a naturally occurring nucleobase (i.e., an A, T, G, C or U)found in at least one naturally occurring nucleic acid (i.e., DNA andRNA), and naturally or non-naturally occurring derivative(s) and analogsof such a nucleobase. A nucleobase generally can form one or morehydrogen bonds (“anneal” or “hybridize”) with at least one naturallyoccurring nucleobase in manner that may substitute for naturallyoccurring nucleobase pairing (e.g., the hydrogen bonding between A andT, G and C, and A and U).

“Purine” and/or “pyrimidine” nucleobase(s) encompass naturally occurringpurine and/or pyrimidine nucleobases and also derivative(s) andanalog(s) thereof, including but not limited to, those a purine orpyrimidine substituted by one or more of an alkyl, caboxyalkyl, amino,hydroxyl, halogen (i.e., fluoro, chloro, bromo, or iodo), thiol oralkylthiol moiety. Preferred alkyl (e.g., alkyl, caboxyalkyl, etc.)moeities comprise of from about 1, about 2, about 3, about 4, about 5,to about 6 carbon atoms. Other non-limiting examples of a purine orpyrimidine include a deazapurine, a 2,6-diaminopurine, a 5-fluorouracil,a xanthine, a hypoxanthine, a 8-bromoguanine, a 8-chloroguanine, abromothymine, a 8-aminoguanine, a 8-hydroxyguanine, a 8-methylguanine, a8-thioguanine, an azaguanine, a 2-aminopurine, a 5-ethylcytosine, a5-methylcyosine, a 5-bromouracil, a 5-ethyluracil, a 5-iodouracil, a5-chlorouracil, a 5-propyluracil, a thiouracil, a 2-methyladenine, amethylthioadenine, a N,N-diemethyladenine, an azaadenines, a8-bromoadenine, a 8-hydroxyadenine, a 6-hydroxyaminopurine, a6-thiopurine, a 4-(6-aminohexyl/cytosine), and the like.

A nucleobase may be comprised in a nucleoside or nucleotide, using anychemical or natural synthesis method described herein or known to one ofordinary skill in the art.

B. Nucleosides

As used herein, a “nucleoside” refers to an individual chemical unitcomprising a nucleobase covalently attached to a nucleobase linkermoiety. A non-limiting example of a “nucleobase linker moiety” is asugar comprising 5-carbon atoms (i.e., a “5-carbon sugar”), includingbut not limited to a deoxyribose, a ribose, an arabinose, or aderivative or an analog of a 5-carbon sugar. Non-limiting examples of aderivative or an analog of a 5-carbon sugar include a2′-fluoro-2′-deoxyribose or a carbocyclic sugar where a carbon issubstituted for an oxygen atom in the sugar ring.

Different types of covalent attachment(s) of a nucleobase to anucleobase linker moiety are known in the art. By way of non-limitingexample, a nucleoside comprising a purine (i.e., A or G) or a7-deazapurine nucleobase typically covalently attaches the 9 position ofa purine or a 7-deazapurine to the 1′-position of a 5-carbon sugar. Inanother non-limiting example, a nucleoside comprising a pyrimidinenucleobase (i.e., C, T or U) typically covalently attaches a 1 positionof a pyrimidine to a 1′-position of a 5-carbon sugar (Kornberg andBaker, 1992).

C. Nucleotides

As used herein, a “nucleotide” refers to a nucleoside further comprisinga “backbone moiety”. A backbone moiety generally covalently attaches anucleotide to another molecule comprising a nucleotide, or to anothernucleotide to form a nucleic acid. The “backbone moiety” in naturallyoccurring nucleotides typically comprises a phosphorus moiety, which iscovalently attached to a 5-carbon sugar. The attachment of the backbonemoiety typically occurs at either the 3′- or 5′-position of the 5-carbonsugar. However, other types of attachments are known in the art,particularly when a nucleotide comprises derivatives or analogs of anaturally occurring 5-carbon sugar or phosphorus moiety.

D. Nucleic Acid Analogs

A nucleic acid may comprise, or be composed entirely of, a derivative oranalog of a nucleobase, a nucleobase linker moiety and/or backbonemoiety that may be present in a naturally occurring nucleic acid. Asused herein a “derivative” refers to a chemically modified or alteredform of a naturally occurring molecule, while the terms “mimic” or“analog” refer to a molecule that may or may not structurally resemble anaturally occurring molecule or moiety, but possesses similar functions.As used herein, a “moiety” generally refers to a smaller chemical ormolecular component of a larger chemical or molecular structure.Nucleobase, nucleoside and nucleotide analogs or derivatives are wellknown in the art, and have been described (see for example, Scheit,1980, incorporated herein by reference).

Additional non-limiting examples of nucleosides, nucleotides or nucleicacids comprising 5-carbon sugar and/or backbone moiety derivatives oranalogs, include those in U.S. Pat. No. 5,681,947 which describesoligonucleotides comprising purine derivatives that form triple helixeswith and/or prevent expression of dsDNA; U.S. Pat. Nos. 5,652,099 and5,763,167 which describe nucleic acids incorporating fluorescent analogsof nucleosides found in DNA or RNA, particularly for use as flourescentnucleic acids probes; U.S. Pat. No. 5,614,617 which describesoligonucleotide analogs with substitutions on pyrimidine rings thatpossess enhanced nuclease stability; U.S. Pat. Nos. 5,670,663, 5,872,232and 5,859,221 which describe oligonucleotide analogs with modified5-carbon sugars (i.e., modified 2′-deoxyfuranosyl moieties) used innucleic acid detection; U.S. Pat. No. 5,446,137 which describesoligonucleotides comprising at least one 5-carbon sugar moietysubstituted at the 4′ position with a substituent other than hydrogenthat can be used in hybridization assays; U.S. Pat. No. 5,886,165 whichdescribes oligonucleotides with both deoxyribonucleotides with 3′-5′internucleotide linkages and ribonucleotides with 2′-5′ internucleotidelinkages; U.S. Pat. No. 5,714,606 which describes a modifiedinternucleotide linkage wherein a 3′-position oxygen of theinternucleotide linkage is replaced by a carbon to enhance the nucleaseresistance of nucleic acids; U.S. Pat. No. 5,672,697 which describesoligonucleotides containing one or more 5′ methylene phosphonateinternucleotide linkages that enhance nuclease resistance; U.S. Pat.Nos. 5,466,786 and 5,792,847 which describe the linkage of a substituentmoiety which may comprise a drug or label to the 2′ carbon of anoligonucleotide to provide enhanced nuclease stability and ability todeliver drugs or detection moieties; U.S. Pat. No. 5,223,618 whichdescribes oligonucleotide analogs with a 2 or 3 carbon backbone linkageattaching the 4′ position and 3′ position of adjacent 5-carbon sugarmoiety to enhanced cellular uptake, resistance to nucleases andhybridization to target RNA; U.S. Pat. No. 5,470,967 which describesoligonucleotides comprising at least one sulfamate or sulfamideinternucleotide linkage that are useful as nucleic acid hybridizationprobe; U.S. Pat. Nos. 5,378,825, 5,777,092, 5,623,070, 5,610,289 and5,602,240 which describe oligonucleotides with three or four atom linkermoiety replacing phosphodiester backbone moiety used for improvednuclease resistance, cellular uptake and regulating RNA expression; U.S.Pat. No. 5,858,988 which describes hydrophobic carrier agent attached tothe 2′-0 position of oligonucleotides to enhanced their membranepermeability and stability; U.S. Pat. No. 5,214,136 which describesoligonucleotides conjugated to anthraquinone at the 5′ terminus thatpossess enhanced hybridization to DNA or RNA; enhanced stability tonucleases; U.S. Pat. No. 5,700,922 which describes PNA-DNA-PNA chimeraswherein the DNA comprises 2′-deoxy-erythro-pentofuranosyl nucleotidesfor enhanced nuclease resistance, binding affinity, and ability toactivate RNase H; and U.S. Pat. No. 5,708,154 which describes RNA linkedto a DNA to form a DNA-RNA hybrid.

E. Polyether and Peptide Nucleic Acids

In certain embodiments, it is contemplated that a nucleic acidcomprising a derivative or analog of a nucleoside or nucleotide may beused in the methods and compositions of the disclosure. A non-limitingexample is a “polyether nucleic acid”, described in U.S. Pat. No.5,908,845, incorporated herein by reference. In a polyether nucleicacid, one or more nucleobases are linked to chiral carbon atoms in apolyether backbone.

Another non-limiting example is a “peptide nucleic acid”, also known asa “PNA”, “peptide-based nucleic acid analog” or “PENAM”, described inU.S. Pat. Nos. 5,786,461, 5,891,625, 5,773,571, 5,766,855, 5,736,336,5,719,262, 5,714,331, 5,539,082, and WO 92/20702, each of which isincorporated herein by reference. Peptide nucleic acids generally haveenhanced sequence specificity, binding properties, and resistance toenzymatic degradation in comparison to molecules such as DNA and RNA(Egholm et al., 1993; PCT/EP/01219). A peptide nucleic acid generallycomprises one or more nucleotides or nucleosides that comprise anucleobase moiety, a nucleobase linker moiety that is not a 5-carbonsugar, and/or a backbone moiety that is not a phosphate backbone moiety.Examples of nucleobase linker moieties described for PNAs include azanitrogen atoms, amido and/or ureido tethers (see for example, U.S. Pat.No. 5,539,082). Examples of backbone moieties described for PNAs includean aminoethylglycine, polyamide, polyethyl, polythioamide,polysulfinamide or polysulfonamide backbone moiety.

In certain embodiments, a nucleic acid analogue such as a peptidenucleic acid may be used to inhibit nucleic acid amplification, such asin PCR, to reduce false positives and discriminate between single basemutants, as described in U.S. Pat. No. 5,891,625. Other modificationsand uses of nucleic acid analogs are known in the art, and areencompassed herein. In a non-limiting example, U.S. Pat. No. 5,786,461describes PNAs with amino acid side chains attached to the PNA backboneto enhance solubility of the molecule. In another example, the cellularuptake property of PNAs is increased by attachment of a lipophilicgroup. U.S. application Ser. No. 117,363 describes several alkylaminomoeities used to enhance cellular uptake of a PNA. Another example isdescribed in U.S. Pat. Nos. 5,766,855, 5,719,262, 5,714,331 and5,736,336, which describe PNAs comprising naturally and non-naturallyoccurring nucleobases and alkylamine side chains that provideimprovements in sequence specificity, solubility and/or binding affinityrelative to a naturally occurring nucleic acid.

F. Preparation of Nucleic Acids

A nucleic acid may be made by any technique known to one of ordinaryskill in the art, such as for example, chemical synthesis, enzymaticproduction or biological production. Non-limiting examples of asynthetic nucleic acid (e.g., a synthetic oligonucleotide), include anucleic acid made by in vitro chemical synthesis using phosphotriester,phosphite or phosphoramidite chemistry and solid phase techniques suchas described in EP 266,032, incorporated herein by reference, or viadeoxynucleoside H-phosphonate intermediates as described by Froehler etal., 1986 and U.S. Pat. No. 5,705,629, each incorporated herein byreference. In the methods of the present disclosure, one or moreoligonucleotide may be used. Various different mechanisms ofoligonucleotide synthesis have been disclosed in for example, U.S. Pat.Nos. 4,659,774, 4,816,571, 5,141,813, 5,264,566, 4,959,463, 5,428,148,5,554,744, 5,574,146, 5,602,244, each of which is incorporated herein byreference.

A non-limiting example of an enzymatically produced nucleic acid includeone produced by enzymes in amplification reactions such as PCR™ (see forexample, U.S. Pat. Nos. 4,683,202 and 4,682,195, each incorporatedherein by reference), or the synthesis of an oligonucleotide describedin U.S. Pat. No. 5,645,897, incorporated herein by reference. Anon-limiting example of a biologically produced nucleic acid includes arecombinant nucleic acid produced (i.e., replicated) in a living cell,such as a recombinant DNA vector replicated in bacteria (see forexample, Sambrook et al. 1989, incorporated herein by reference).

G. Purification of Nucleic Acids

A nucleic acid may be purified on polyacrylamide gels, cesium chloridecentrifugation gradients, or by any other means known to one of ordinaryskill in the art (see for example, Sambrook et al., 1989, incorporatedherein by reference).

In certain aspect, the present disclosure concerns a nucleic acid thatis an isolated nucleic acid. As used herein, the term “isolated nucleicacid” refers to a nucleic acid molecule (e.g., an RNA or DNA molecule)that has been isolated free of, or is otherwise free of, the bulk of thetotal genomic and transcribed nucleic acids of one or more cells. Incertain embodiments, “isolated nucleic acid” refers to a nucleic acidthat has been isolated free of, or is otherwise free of, bulk ofcellular components or in vitro reaction components such as for example,macromolecules such as lipids or proteins, small biological molecules,and the like.

H. Nucleic Acid Segments

In certain embodiments, the nucleic acid is a nucleic acid segment. Asused herein, the term “nucleic acid segment,” are smaller fragments of anucleic acid, such as for non-limiting example, those that encode onlypart of the peptide or polypeptide sequence. Thus, a “nucleic acidsegment” may comprise any part of a gene sequence, of from about 2nucleotides to the full length of the peptide or polypeptide encodingregion.

Various nucleic acid segments may be designed based on a particularnucleic acid sequence, and may be of any length. By assigning numericvalues to a sequence, for example, the first residue is 1, the secondresidue is 2, etc., an algorithm defining all nucleic acid segments canbe created:

n to n+y

where n is an integer from 1 to the last number of the sequence and y isthe length of the nucleic acid segment minus one, where n+y does notexceed the last number of the sequence. Thus, for a 10-mer, the nucleicacid segments correspond to bases 1 to 10, 2 to 11, 3 to 12 . . . and soon. For a 15-mer, the nucleic acid segments correspond to bases 1 to 15,2 to 16, 3 to 17 . . . and so on. For a 20-mer, the nucleic segmentscorrespond to bases 1 to 20, 2 to 21, 3 to 22 . . . and so on. Incertain embodiments, the nucleic acid segment may be a probe or primer.As used herein, a “probe” generally refers to a nucleic acid used in adetection method or composition. As used herein, a “primer” generallyrefers to a nucleic acid used in an extension or amplification method orcomposition.

I. Nucleic Acid Complements

The present disclosure also encompasses a nucleic acid that iscomplementary to a p63 nucleic acid. In particular embodiments thedisclosure encompasses a nucleic acid or a nucleic acid segmentcomplementary to the p63 encoding sequence. A nucleic acid“complement(s)” or is “complementary” to another nucleic acid when it iscapable of base-pairing with another nucleic acid according to thestandard Watson-Crick, Hoogsteen or reverse Hoogsteen bindingcomplementarity rules. As used herein “another nucleic acid” may referto a separate molecule or a spatial separated sequence of the samemolecule.

As used herein, the term “complementary” or “complement(s)” also refersto a nucleic acid comprising a sequence of consecutive nucleobases orsemiconsecutive nucleobases (e.g., one or more nucleobase moieties arenot present in the molecule) capable of hybridizing to another nucleicacid strand or duplex even if less than all the nucleobases do not basepair with a counterpart nucleobase. In certain embodiments, a“complementary” nucleic acid comprises a sequence in which about 70%,about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about77%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%,about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 98%, about 99%, to about 100%, and any rangederivable therein, of the nucleobase sequence is capable of base-pairingwith a single or double stranded nucleic acid molecule duringhybridization. In certain embodiments, the term “complementary” refersto a nucleic acid that may hybridize to another nucleic acid strand orduplex in stringent conditions, as would be understood by one ofordinary skill in the art.

In certain embodiments, a “partly complementary” nucleic acid comprisesa sequence that may hybridize in low stringency conditions to a singleor double stranded nucleic acid, or contains a sequence in which lessthan about 70% of the nucleobase sequence is capable of base-pairingwith a single or double stranded nucleic acid molecule duringhybridization.

J. Hybridization

As used herein, “hybridization”, “hybridizes” or “capable ofhybridizing” is understood to mean the forming of a double or triplestranded molecule or a molecule with partial double or triple strandednature. The term “anneal” as used herein is synonymous with “hybridize.”The term “hybridization”, “hybridize(s)” or “capable of hybridizing”encompasses the terms “stringent condition(s)” or “high stringency” andthe terms “low stringency” or “low stringency condition(s).”

As used herein “stringent condition(s)” or “high stringency” are thoseconditions that allow hybridization between or within one or morenucleic acid strand(s) containing complementary sequence(s), butprecludes hybridization of random sequences. Stringent conditionstolerate little, if any, mismatch between a nucleic acid and a targetstrand. Such conditions are well known to those of ordinary skill in theart, and are preferred for applications requiring high selectivity.Non-limiting applications include isolating a nucleic acid, such as agene or a nucleic acid segment thereof, or detecting at least onespecific mRNA transcript or a nucleic acid segment thereof, and thelike.

Stringent conditions may comprise low salt and/or high temperatureconditions, such as provided by about 0.02 M to about 0.15 M NaCl attemperatures of about 50° C. to about 70° C. It is understood that thetemperature and ionic strength of a desired stringency are determined inpart by the length of the particular nucleic acid(s), the length andnucleobase content of the target sequence(s), the charge composition ofthe nucleic acid(s), and to the presence or concentration of formamide,tetramethylammonium chloride or other solvent(s) in a hybridizationmixture.

It is also understood that these ranges, compositions and conditions forhybridization are mentioned by way of non-limiting examples only, andthat the desired stringency for a particular hybridization reaction isoften determined empirically by comparison to one or more positive ornegative controls. Depending on the application envisioned it ispreferred to employ varying conditions of hybridization to achievevarying degrees of selectivity of a nucleic acid towards a targetsequence. In a non-limiting example, identification or isolation of arelated target nucleic acid that does not hybridize to a nucleic acidunder stringent conditions may be achieved by hybridization at lowtemperature and/or high ionic strength. Such conditions are termed “lowstringency” or “low stringency conditions”, and non-limiting examples oflow stringency include hybridization performed at about 0.15 M to about0.9 M NaCl at a temperature range of about 20° C. to about 50° C. Ofcourse, it is within the skill of one in the art to further modify thelow or high stringency conditions to suite a particular application.

As used herein “wild-type” refers to the naturally occurring sequence ofa nucleic acid at a genetic locus in the genome of an organism, or asequence transcribed or translated from such a nucleic acid. Thus, theterm “wild-type” also may refer to an amino acid sequence encoded by anucleic acid. As a genetic locus may have more than one sequence oralleles in a population of individuals, the term “wild-type” encompassesall such naturally occurring allele(s). As used herein the term“polymorphic” means that variation exists (i.e., two or more allelesexist) at a genetic locus in the individuals of a population. As usedherein “mutant” refers to a change in the sequence of a nucleic acid orits encoded protein, polypeptide or peptide that is the result of thehand of man.

The present disclosure also concerns the isolation or creation of arecombinant construct or a recombinant host cell through the applicationof recombinant nucleic acid technology known to those of skill in theart or as described herein. A recombinant construct or host cell maycomprise a nucleic acid, and may express a protein, peptide or peptide,or at least one biologically functional equivalent thereof.

Herein, in certain embodiments, a “gene” refers to a nucleic acid thatis transcribed. In certain aspects, the gene includes regulatorysequences involved in transcription, or message production orcomposition. In particular embodiments, the gene comprises transcribedsequences that encode for a protein, polypeptide or peptide. As will beunderstood by those in the art, this function term “gene” includes bothgenomic sequences, RNA or cDNA sequences or smaller engineered nucleicacid segments, including nucleic acid segments of a non-transcribed partof a gene, including but not limited to the non-transcribed promoter orenhancer regions of a gene. Smaller engineered gene nucleic acidsegments may express, or may be adapted to express using nucleic acidmanipulation technology, proteins, polypeptides, domains, peptides,fusion proteins, mutants and/or such like.

“Isolated substantially away from other coding sequences” means that thegene of interest forms the significant part of the coding region of thenucleic acid, or that the nucleic acid does not contain large portionsof naturally-occurring coding nucleic acids, such as large chromosomalfragments, other functional genes, RNA or cDNA coding regions. Ofcourse, this refers to the nucleic acid as originally isolated, and doesnot exclude genes or coding regions later added to the nucleic acid bythe hand of man.

The nucleic acid(s) of the present disclosure, regardless of the lengthof the sequence itself, may be combined with other nucleic acidsequences, including but not limited to, promoters, enhancers,polyadenylation signals, restriction enzyme sites, multiple cloningsites, coding segments, and the like, to create one or more nucleic acidconstruct(s). As used herein, a “nucleic acid construct” is a nucleicacid engineered or altered by the hand of man, and generally comprisesone or more nucleic acid sequences organized by the hand of man.

In a non-limiting example, one or more nucleic acid constructs may beprepared that include a contiguous stretch of nucleotides identical toor complementary (at least in part) to p63. A nucleic acid construct maybe about 3, about 5, about 8, about 10 to about 14, or about 15, about20, about 30, about 40, about 50, about 100, about 115, about 200, about500, about 600, or about 650 nucleotides in length, as well asconstructs of greater size, up to and including vector sizes (includingall intermediate lengths and intermediate ranges. It will be readilyunderstood that “intermediate lengths” and “intermediate ranges”, asused herein, means any length or range including or between the quotedvalues (i.e., all integers including and between such values).Non-limiting examples of intermediate lengths include about 11, about12, about 13, about 16, about 17, about 18, about 19, etc.; about 21,about 22, about 23, etc.; about 31, about 32, etc.; about 51, about 52,about 53, etc.; about 101, about 102, about 103, etc.; about 151, about152, about 153, etc.; about 600, about 601, about 605, about 610, etc.etc., etc. Non-limiting examples of intermediate ranges include about 3to about 32, about 150 to about 750, etc.

In certain embodiments, the nucleic acid construct is a recombinantvector. In particular embodiments, the disclosure concerns one or morerecombinant vector(s) comprising nucleic acid sequences that encode anHand2 or myocardin protein, polypeptide or peptide. In particularaspects, the recombinant vectors are DNA vectors.

The term “biologically functional equivalent” is well understood in theart and is further defined in detail herein. Accordingly, a sequencethat has 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% of amino acids that are identical or functionallyequivalent to the amino acids encoded by the Hand2 and myocardin nucleicacids, respectively, provided the biological activity of the protein,polypeptide or peptide is maintained.

In certain other embodiments, the disclosure concerns at least onerecombinant vector that includes within its sequence a nucleic acidsequence the expresses p63 shRNA. In specific embodiments, thedisclosure concerns at least one recombinant vector that includes withinits sequence a nucleic acid sequence that expresses Hand2 nucleic acid.In another embodiment, there is at least one recombinant vector thatincludes within its sequence a nucleic acid sequence that expressesmyocardin nucleic acid.

The term “functionally equivalent codon” is used herein to refer tocodons that encode the same amino acid, such as the six codons forarginine and serine, and also refers to codons that encode biologicallyequivalent amino acids. Codon usage for various organisms and organellescan be found in the literature. Thus, it is contemplated that codonusage may be optimized for other animals, as well as other organismssuch as a prokaryote (e.g., an eubacteria, an archaea), an eukaryote(e.g., a protist, a plant, a fungi, an animal), a virus and the like, aswell as organelles that contain nucleic acids, such as mitochondria,chloroplasts and the like, based on the preferred codon usage as wouldbe known to those of ordinary skill in the art.

It will also be understood that amino acid sequences or nucleic acidsequences may include additional residues, such as additional N- orC-terminal amino acids or 5′ or 3′ sequences, or various combinationsthereof, and yet still be essentially as set forth in one of thesequences disclosed herein, so long as the sequence meets the criteriaset forth above, including the maintenance of biological protein,polypeptide or peptide activity where expression of a proteinaceouscomposition is concerned. The addition of terminal sequencesparticularly applies to nucleic acid sequences that may, for example,include various non-coding sequences flanking either of the 5′ and/or 3′portions of the coding region or may include various internal sequences,i.e., introns, which are known to occur within genes.

Excepting intronic and flanking regions, and allowing for the degeneracyof the genetic code, nucleic acid sequences that have between about 70%and about 79%; or more preferably, between about 80% and about 89%; oreven more particularly, between about 90% and about 99%; of nucleotidesthat are identical to the nucleotides of the noted GenBank® sequencesdisclosed herein are encompassed in the disclosure.

Recombinant vectors and isolated nucleic acid segments may thereforevariously include Hand2 or myocardin coding regions themselves, codingregions bearing selected alterations or modifications in the basiccoding region, and they may encode larger polypeptides or peptides thatnevertheless include such coding regions or may encode biologicallyfunctional equivalent proteins, polypeptide or peptides that havevariant amino acids sequences.

The nucleic acids of the present disclosure may encompass biologicallyfunctional equivalent coding sequences for Hand2 or myocardin proteins,polypeptides, or peptides. Such sequences may arise as a consequence ofcodon redundancy or functional equivalency that are known to occurnaturally within nucleic acid sequences or the proteins, polypeptides orpeptides thus encoded. Alternatively, functionally equivalent proteins,polypeptides or peptides may be created via the application ofrecombinant DNA technology, in which changes in the protein, polypeptideor peptide structure may be engineered, based on considerations of theproperties of the amino acids being exchanged. Changes designed by manmay be introduced, for example, through the application of site-directedmutagenesis techniques as discussed herein below, e.g., to introduceimprovements or alterations to the antigenicity of the protein,polypeptide or peptide, or to test mutants in order to examine protein,polypeptide or peptide activity at the molecular level.

IV. Nucleic Acid-Based Expression Systems

In particular embodiments of the disclosure, the p63, p53, and/orp21-inactivating agents (such as siRNA or shRNA nucleic acid), in somecases one or more cardiac cell reprogramming factors (such as Hand2and/or myocardin), and in some cases one or more destabilizing agentsand/or anti-fibrotic agents and/or angiogenic factors are provided innucleic acid form to an individual in need thereof. Although in somecases the nucleic acids are not comprised on a vector, in particularembodiments the nucleic acids are present on one or more vectors. Inparticular embodiments, the different nucleic acids are present on thesame vector, whereas in other cases they are present on two or threeseparate vectors. The vectors may be viral or non-viral in nature. FIG.25 provides an illustration of an embodiment of a vector for use inmethods of the present disclosure (see also Mathison et al., J ThoracCardiovasc Surg. 2014 October; 148(4):1656-1664).

The vectors utilized in the embodiments of the disclosure may have oneor more means for targeted delivery to cardiac tissue and/or targetedexpression in certain cells. In some cases the vector(s) are provided tothe individual with localized delivery to the heart, whereas in othercases the vectors are provided systemically to the individual with ameans for targeted delivery to cardiac tissue and/or targeted expressionin certain cells, such as cardiac fibroblasts, for example. In certainembodiments, the p63, p53, and/or p21 shRNA or siRNA and one or both ofcardiac cell reprogramming factors (such as Hand2 and myocardin)polynucleotides are on the same molecule, although in some embodimentsthe p63, p53, and/or p21 shRNA or siRNA and one or both of cardiac cellreprogramming factors polynucleotides are on different molecules. Whenthe shRNA and one or both of cardiac cell reprogramming factors areexpressed from the same polynucleotide, they may have the same ordifferent regulatory regions for their expression. In specificembodiments, the p63, p53, and/or p21-inactivating agent polynucleotidesand one or more chromatin destabilizing agent polynucleotides are on thesame or different molecules.

In particular embodiments, an expression vector for use in thedisclosure may comprise one or more suitable restriction enzymedigestion sequences, start codons, stop codons, nuclear localizationsignals, protease cutting codons, selectable markers, origins ofreplication, regulatory regions, multiple cloning sites, and acombination thereof. Such moieties may be positioned in the expressionvector in any suitable order.

A. Vectors

The term “vector” is used to refer to a carrier nucleic acid moleculeinto which a nucleic acid sequence can be inserted for introduction intoa cell where it can be replicated. A nucleic acid sequence can be“exogenous,” which means that it is foreign to the cell into which thevector is being introduced or that the sequence is homologous to asequence in the cell but in a position within the host cell nucleic acidin which the sequence is ordinarily not found. Vectors include plasmids,cosmids, viruses (bacteriophage, animal viruses, and plant viruses), andartificial chromosomes (e.g., YACs). One of skill in the art would bewell equipped to construct a vector through standard recombinanttechniques (see, for example, Maniatis et al., 1988 and Ausubel et al.,1994, both incorporated herein by reference).

The term “expression vector” refers to any type of genetic constructcomprising a nucleic acid coding for a RNA capable of being transcribed.In some cases, RNA molecules are then translated into a protein,polypeptide, or peptide. In other cases, these sequences are nottranslated, for example, in the production of antisense molecules orribozymes. Expression vectors can contain a variety of “controlsequences,” which refer to nucleic acid sequences necessary for thetranscription and possibly translation of an operably linked codingsequence in a particular host cell. In addition to control sequencesthat govern transcription and translation, vectors and expressionvectors may contain nucleic acid sequences that serve other functions aswell and are described infra.

1. Promoters and Enhancers

A “promoter” is a control sequence that is a region of a nucleic acidsequence at which initiation and rate of transcription are controlled.It may contain genetic elements at which regulatory proteins andmolecules may bind, such as RNA polymerase and other transcriptionfactors, to initiate the specific transcription a nucleic acid sequence.The phrases “operatively positioned,” “operatively linked,” “undercontrol,” and “under transcriptional control” mean that a promoter is ina correct functional location and/or orientation in relation to anucleic acid sequence to control transcriptional initiation and/orexpression of that sequence.

In embodiments of the disclosure, a CMV promoter or a tissue-specificpromoter may be employed. The tissue-specific promoter may be a cardiactissue specific promoter. Examples of cardiac tissue specific promotersinclude ventricle-specific myosin light chain-2 (mlc-2v); alpha-myosinheavy chain (α-MHC). In specific embodiments, a fibroblast-specificpromoter is employed, such as Fsp1 promoter.

A promoter generally comprises a sequence that functions to position thestart site for RNA synthesis. The best known example of this is the TATAbox, but in some promoters lacking a TATA box, such as, for example, thepromoter for the mammalian terminal deoxynucleotidyl transferase geneand the promoter for the SV40 late genes, a discrete element overlyingthe start site itself helps to fix the place of initiation. Additionalpromoter elements regulate the frequency of transcriptional initiation.Typically, these are located in the region 30-110 bp upstream of thestart site, although a number of promoters have been shown to containfunctional elements downstream of the start site as well. To bring acoding sequence “under the control of” a promoter, one positions the 5′end of the transcription initiation site of the transcriptional readingframe “downstream” of (i.e., 3′ of) the chosen promoter. The “upstream”promoter stimulates transcription of the DNA and promotes expression ofthe encoded RNA.

The spacing between promoter elements frequently is flexible, so thatpromoter function is preserved when elements are inverted or movedrelative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either cooperatively or independently to activatetranscription. A promoter may or may not be used in conjunction with an“enhancer,” which refers to a cis-acting regulatory sequence involved inthe transcriptional activation of a nucleic acid sequence.

A promoter may be one naturally associated with a nucleic acid sequence,as may be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment and/or exon. Such a promoter can bereferred to as “endogenous.” Similarly, an enhancer may be one naturallyassociated with a nucleic acid sequence, located either downstream orupstream of that sequence. Alternatively, certain advantages will begained by positioning the coding nucleic acid segment under the controlof a recombinant or heterologous promoter, which refers to a promoterthat is not normally associated with a nucleic acid sequence in itsnatural environment. A recombinant or heterologous enhancer refers alsoto an enhancer not normally associated with a nucleic acid sequence inits natural environment. Such promoters or enhancers may includepromoters or enhancers of other genes, and promoters or enhancersisolated from any other virus, or prokaryotic or eukaryotic cell, andpromoters or enhancers not “naturally occurring,” i.e., containingdifferent elements of different transcriptional regulatory regions,and/or mutations that alter expression. For example, promoters that aremost commonly used in recombinant DNA construction include theβ-lactamase (penicillinase), lactose and tryptophan (trp) promotersystems. In addition to producing nucleic acid sequences of promotersand enhancers synthetically, sequences may be produced using recombinantcloning and/or nucleic acid amplification technology, including PCR™, inconnection with the compositions disclosed herein (see U.S. Pat. Nos.4,683,202 and 5,928,906, each incorporated herein by reference).Furthermore, it is contemplated the control sequences that directtranscription and/or expression of sequences within non-nuclearorganelles such as mitochondria, chloroplasts, and the like, can beemployed as well.

Naturally, it will be important to employ a promoter and/or enhancerthat effectively directs the expression of the DNA segment in theorganelle, cell type, tissue, organ, or organism chosen for expression.Those of skill in the art of molecular biology generally know the use ofpromoters, enhancers, and cell type combinations for protein expression,(see, for example Sambrook et al. 1989, incorporated herein byreference). The promoters employed may be constitutive, tissue-specific,inducible, and/or useful under the appropriate conditions to direct highlevel expression of the introduced DNA segment, such as is advantageousin the large-scale production of recombinant proteins and/or peptides.The promoter may be heterologous or endogenous.

Additionally any promoter/enhancer combination (as per, for example, theEukaryotic Promoter Data Base EPDB, http://www.epd.isb-sib.ch/) couldalso be used to drive expression. Use of a T3, T7 or SP6 cytoplasmicexpression system is another possible embodiment. Eukaryotic cells cansupport cytoplasmic transcription from certain bacterial promoters ifthe appropriate bacterial polymerase is provided, either as part of thedelivery complex or as an additional genetic expression construct.

2. Initiation Signals and Internal Ribosome Binding Sites

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be “in-frame” with the reading frame of thedesired coding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancerelements.

In certain embodiments of the disclosure, the use of internal ribosomeentry sites (IRES) elements are used to create multigene, orpolycistronic, messages. IRES elements are able to bypass the ribosomescanning model of 5′ methylated Cap dependent translation and begintranslation at internal sites (Pelletier and Sonenberg, 1988). IRESelements from two members of the picornavirus family (polio andencephalomyocarditis) have been described (Pelletier and Sonenberg,1988), as well an IRES from a mammalian message (Macejak and Sarnow,1991). IRES elements can be linked to heterologous open reading frames.Multiple open reading frames can be transcribed together, each separatedby an IRES, creating polycistronic messages. By virtue of the IRESelement, each open reading frame is accessible to ribosomes forefficient translation. Multiple genes can be efficiently expressed usinga single promoter/enhancer to transcribe a single message (see U.S. Pat.Nos. 5,925,565 and 5,935,819, each herein incorporated by reference).

3. Multiple Cloning Sites

Vectors can include a multiple cloning site (MCS), which is a nucleicacid region that contains multiple restriction enzyme sites, any ofwhich can be used in conjunction with standard recombinant technology todigest the vector (see, for example, Carbonelli et al., 1999, Levensonet al., 1998, and Cocea, 1997, incorporated herein by reference.)“Restriction enzyme digestion” refers to catalytic cleavage of a nucleicacid molecule with an enzyme that functions only at specific locationsin a nucleic acid molecule. Many of these restriction enzymes arecommercially available. Use of such enzymes is widely understood bythose of skill in the art. Frequently, a vector is linearized orfragmented using a restriction enzyme that cuts within the MCS to enableexogenous sequences to be ligated to the vector. “Ligation” refers tothe process of forming phosphodiester bonds between two nucleic acidfragments, which may or may not be contiguous with each other.Techniques involving restriction enzymes and ligation reactions are wellknown to those of skill in the art of recombinant technology.

4. Splicing Sites

Most transcribed eukaryotic RNA molecules will undergo RNA splicing toremove introns from the primary transcripts. Vectors containing genomiceukaryotic sequences may require donor and/or acceptor splicing sites toensure proper processing of the transcript for protein expression (see,for example, Chandler et al., 1997, herein incorporated by reference.)

5. Termination Signals

The vectors or constructs of the present disclosure will generallycomprise at least one termination signal. A “termination signal” or“terminator” is comprised of the DNA sequences involved in specifictermination of an RNA transcript by an RNA polymerase. Thus, in certainembodiments a termination signal that ends the production of an RNAtranscript is contemplated. A terminator may be necessary in vivo toachieve desirable message levels.

In eukaryotic systems, the terminator region may also comprise specificDNA sequences that permit site-specific cleavage of the new transcriptso as to expose a polyadenylation site. This signals a specializedendogenous polymerase to add a stretch of about 200 A residues (polyA)to the 3′ end of the transcript. RNA molecules modified with this polyAtail appear to more stable and are translated more efficiently. Thus, inother embodiments involving eukaryotes, it is preferred that thatterminator comprises a signal for the cleavage of the RNA, and it ismore preferred that the terminator signal promotes polyadenylation ofthe message. The terminator and/or polyadenylation site elements canserve to enhance message levels and to minimize read through from thecassette into other sequences.

Terminators contemplated for use in the disclosure include any knownterminator of transcription described herein or known to one of ordinaryskill in the art, including but not limited to, for example, thetermination sequences of genes, such as for example the bovine growthhormone terminator or viral termination sequences, such as for examplethe SV40 terminator. In certain embodiments, the termination signal maybe a lack of transcribable or translatable sequence, such as due to asequence truncation.

6. Polyadenylation Signals

In expression, particularly eukaryotic expression, one will typicallyinclude a polyadenylation signal to effect proper polyadenylation of thetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the disclosure, and any suchsequence may be employed. Preferred embodiments include the SV40polyadenylation signal or the bovine growth hormone polyadenylationsignal, convenient and known to function well in various target cells.Polyadenylation may increase the stability of the transcript or mayfacilitate cytoplasmic transport.

7. Origins of Replication

In order to propagate a vector in a host cell, it may contain one ormore origins of replication sites (often termed “ori”), which is aspecific nucleic acid sequence at which replication is initiated.Alternatively an autonomously replicating sequence (ARS) can be employedif the host cell is yeast.

8. Selectable and Screenable Markers

In certain embodiments of the disclosure, cells containing a nucleicacid construct of the present disclosure may be identified in vitro orin vivo by including a marker in the expression vector. Such markerswould confer an identifiable change to the cell permitting easyidentification of cells containing the expression vector. Generally, aselectable marker is one that confers a property that allows forselection. A positive selectable marker is one in which the presence ofthe marker allows for its selection, while a negative selectable markeris one in which its presence prevents its selection. An example of apositive selectable marker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning andidentification of transformants, for example, genes that conferresistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin andhistidinol are useful selectable markers. In addition to markersconferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as GFP, whose basis iscolorimetric analysis, are also contemplated. Alternatively, screenableenzymes such as herpes simplex virus thymidine kinase (tk) orchloramphenicol acetyltransferase (CAT) may be utilized. One of skill inthe art would also know how to employ immunologic markers, possibly inconjunction with FACS analysis. The marker used is not believed to beimportant, so long as it is capable of being expressed simultaneouslywith the nucleic acid encoding a gene product. Further examples ofselectable and screenable markers are well known to one of skill in theart.

B. Plasmid Vectors

In certain embodiments, a plasmid vector is contemplated for use totransform a host cell. In general, plasmid vectors containing repliconand control sequences which are derived from species compatible with thehost cell are used in connection with these hosts. The vector ordinarilycarries a replication site, as well as marking sequences which arecapable of providing phenotypic selection in transformed cells. In anon-limiting example, E. coli is often transformed using derivatives ofpBR322, a plasmid derived from an E. coli species. pBR322 contains genesfor ampicillin and tetracycline resistance and thus provides easy meansfor identifying transformed cells. The pBR plasmid, or other microbialplasmid or phage must also contain, or be modified to contain, forexample, promoters which can be used by the microbial organism forexpression of its own proteins.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example, thephage lambda GEM™-11 may be utilized in making a recombinant phagevector which can be used to transform host cells, such as, for example,E. coli LE392.

Genomic integrated plasmids, such as piggybac or sleeping beautytransposon gene delivery plasmids, may be employed for long termtransgenic expression of a nucleic acid in heart or other organ.

Further useful plasmid vectors include pIN vectors (Inouye et al.,1985); and pGEX vectors, for use in generating glutathione S-transferase(GST) soluble fusion proteins for later purification and separation orcleavage. Other suitable fusion proteins are those with β-galactosidase,ubiquitin, and the like.

Bacterial host cells, for example, E. coli, comprising the expressionvector, are grown in any of a number of suitable media, for example, LB.The expression of the recombinant protein in certain vectors may beinduced, as would be understood by those of skill in the art, bycontacting a host cell with an agent specific for certain promoters,e.g., by adding IPTG to the media or by switching incubation to a highertemperature. After culturing the bacteria for a further period,generally of between 2 and 24 h, the cells are collected bycentrifugation and washed to remove residual media.

C. Viral Vectors

The ability of certain viruses to infect cells or enter cells viareceptor-mediated endocytosis, and to integrate into host cell genomeand express viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign nucleic acids into cells (e.g.,mammalian cells). Non-limiting examples of virus vectors that may beused to deliver a nucleic acid of the present disclosure are describedbelow.

1. Adenoviral Vectors

A particular method for delivery of the nucleic acid involves the use ofan adenovirus expression vector. Although adenovirus vectors are knownto have a low capacity for integration into genomic DNA, this feature iscounterbalanced by the high efficiency of gene transfer afforded bythese vectors. “Adenovirus expression vector” is meant to include thoseconstructs containing adenovirus sequences sufficient to (a) supportpackaging of the construct and (b) to ultimately express a tissue orcell-specific construct that has been cloned therein. Knowledge of thegenetic organization or adenovirus, a 36 kb, linear, double-stranded DNAvirus, allows substitution of large pieces of adenoviral DNA withforeign sequences up to 7 kb (Grunhaus and Horwitz, 1992).

2. AAV Vectors

The nucleic acid may be introduced into the cell using adenovirusassisted transfection. Increased transfection efficiencies have beenreported in cell systems using adenovirus coupled systems (Kelleher andVos, 1994; Cotten et al., 1992; Curiel, 1994). Adeno-associated virus(AAV) is an attractive vector system for use in embodiments of thepresent disclosure as it has a high frequency of integration and it caninfect nondividing cells, thus making it useful for delivery of genesinto mammalian cells, for example, in tissue culture (Muzyczka, 1992) orin vivo. AAV has a broad host range for infectivity (Tratschin et al.,1984; Laughlin et al., 1986; Lebkowski et al., 1988; McLaughlin et al.,1988). Details concerning the generation and use of rAAV vectors aredescribed in U.S. Pat. Nos. 5,139,941 and 4,797,368, each incorporatedherein by reference.

3. Retroviral Vectors

Retroviruses have promise as delivery vectors due to their ability tointegrate their genes into the host genome, transferring a large amountof foreign genetic material, infecting a broad spectrum of species andcell types and of being packaged in special cell-lines (Miller, 1992).

In order to construct a retroviral vector, a nucleic acid is insertedinto the viral genome in the place of certain viral sequences to producea virus that is replication-defective. In order to produce virions, apackaging cell line containing the gag, pol, and env genes but withoutthe LTR and packaging components is constructed (Mann et al., 1983).When a recombinant plasmid containing a cDNA, together with theretroviral LTR and packaging sequences is introduced into a special cellline (e.g., by calcium phosphate precipitation for example), thepackaging sequence allows the RNA transcript of the recombinant plasmidto be packaged into viral particles, which are then secreted into theculture media (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al.,1983). The media containing the recombinant retroviruses is thencollected, optionally concentrated, and used for gene transfer.Retroviral vectors are able to infect a broad variety of cell types.However, integration and stable expression require the division of hostcells (Paskind et al., 1975).

Lentiviruses are complex retroviruses, which, in addition to the commonretroviral genes gag, pol, and env, contain other genes with regulatoryor structural function. Lentiviral vectors are well known in the art(see, for example, Naldini et al., 1996; Zufferey et al., 1997; Blomeret al., 1997; U.S. Pat. Nos. 6,013,516 and 5,994,136). Some examples oflentivirus include the Human Immunodeficiency Viruses: HIV-1, HIV-2 andthe Simian Immunodeficiency Virus: SIV. Lentiviral vectors have beengenerated by multiply attenuating the HIV virulence genes, for example,the genes env, vif, vpr, vpu and nef are deleted making the vectorbiologically safe.

Recombinant lentiviral vectors are capable of infecting non-dividingcells and can be used for both in vivo and ex vivo gene transfer andexpression of nucleic acid sequences. For example, recombinantlentivirus capable of infecting a non-dividing cell wherein a suitablehost cell is transfected with two or more vectors carrying the packagingfunctions, namely gag, pol and env, as well as rev and tat is describedin U.S. Pat. No. 5,994,136, incorporated herein by reference. One maytarget the recombinant virus by linkage of the envelope protein with anantibody or a particular ligand for targeting to a receptor of aparticular cell-type. By inserting a sequence (including a regulatoryregion) of interest into the viral vector, along with another gene whichencodes the ligand for a receptor on a specific target cell, forexample, the vector is now target-specific.

4. Other Viral Vectors

Other viral vectors may be employed as vaccine constructs in the presentdisclosure. Vectors derived from viruses such as vaccinia virus(Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988),sindbis virus, cytomegalovirus and herpes simplex virus may be employed.They offer several attractive features for various mammalian cells(Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar etal., 1988; Horwich et al., 1990).

D. Delivery Using Modified Viruses

A nucleic acid to be delivered may be housed within an infective virusthat has been engineered to express a specific binding ligand. The virusparticle will thus bind specifically to the cognate receptors of thetarget cell and deliver the contents to the cell. A novel approachdesigned to allow specific targeting of retrovirus vectors was developedbased on the chemical modification of a retrovirus by the chemicaladdition of lactose residues to the viral envelope. This modificationcan permit the specific infection of hepatocytes via sialoglycoproteinreceptors.

Another approach to targeting of recombinant retroviruses was designedin which biotinylated antibodies against a retroviral envelope proteinand against a specific cell receptor were used. The antibodies werecoupled via the biotin components by using streptavidin (Roux et al.,1989). Using antibodies against major histocompatibility complex class Iand class II antigens, they demonstrated the infection of a variety ofhuman cells that bore those surface antigens with an ecotropic virus invitro (Roux et al., 1989).

E. Vector Delivery and Cell Transformation

Suitable methods for nucleic acid delivery for transformation of anorganelle, a cell, a tissue or an organism for use with the currentdisclosure are believed to include virtually any method by which anucleic acid (e.g., DNA) can be introduced into an organelle, a cell, atissue or an organism, as described herein or as would be known to oneof ordinary skill in the art. Such methods include, but are not limitedto, direct delivery of DNA such as by ex vivo transfection (Wilson etal., 1989, Nabel et al, 1989), by injection (U.S. Pat. Nos. 5,994,624,5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610,5,589,466 and 5,580,859, each incorporated herein by reference),including microinjection (Harlan and Weintraub, 1985; U.S. Pat. No.5,789,215, incorporated herein by reference); by electroporation (U.S.Pat. No. 5,384,253, incorporated herein by reference; Tur-Kaspa et al.,1986; Potter et al., 1984); by calcium phosphate precipitation (Grahamand Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); byusing DEAE-dextran followed by polyethylene glycol (Gopal, 1985); bydirect sonic loading (Fechheimer et al., 1987); by liposome mediatedtransfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau etal., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991)and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988);by microprojectile bombardment (PCT Application Nos. WO 94/09699 and95/06128; U.S. Pat. Nos. 5,610,042; 5,322,783 5,563,055, 5,550,318,5,538,877 and 5,538,880, and each incorporated herein by reference); byagitation with silicon carbide fibers (Kaeppler et al., 1990; U.S. Pat.Nos. 5,302,523 and 5,464,765, each incorporated herein by reference); byAgrobacterium-mediated transformation (U.S. Pat. Nos. 5,591,616 and5,563,055, each incorporated herein by reference); by PEG-mediatedtransformation of protoplasts (Omirulleh et al., 1993; U.S. Pat. Nos.4,684,611 and 4,952,500, each incorporated herein by reference); bydesiccation/inhibition-mediated DNA uptake (Potrykus et al., 1985), andany combination of such methods. Through the application of techniquessuch as these, organelle(s), cell(s), tissue(s) or organism(s) may bestably or transiently transformed.

1. Ex Vivo Transformation

Methods for transfecting vascular cells and tissues removed from anorganism in an ex vivo setting are known to those of skill in the art.For example, canine endothelial cells have been genetically altered byretroviral gene transfer in vitro and transplanted into a canine (Wilsonet al., 1989). In another example, yucatan minipig endothelial cellswere tranfected by retrovirus in vitro and transplanted into an arteryusing a double-balloon catheter (Nabel et al., 1989). Thus, it iscontemplated that cells or tissues may be removed and tranfected ex vivousing the nucleic acids of the present disclosure. In particularaspects, the transplanted cells or tissues may be placed into anorganism. In preferred facets, a nucleic acid is expressed in thetransplanted cells or tissues.

2. Injection

In certain embodiments, a nucleic acid may be delivered to an organelle,a cell, a tissue or an organism via one or more injections (i.e., aneedle injection), such as, for example, subcutaneously, intradermally,intramuscularly, intervenously, intraperitoneally, etc. Methods ofinjection are well known to those of ordinary skill in the art (e.g.,injection of a composition comprising a saline solution). Furtherembodiments of the present disclosure include the introduction of anucleic acid by direct microinjection.

3. Electroporation

In certain embodiments of the present disclosure, a nucleic acid isintroduced into an organelle, a cell, a tissue or an organism viaelectroporation. Electroporation involves the exposure of a suspensionof cells and DNA to a high-voltage electric discharge. In some variantsof this method, certain cell wall-degrading enzymes, such aspectin-degrading enzymes, are employed to render the target recipientcells more susceptible to transformation by electroporation thanuntreated cells (U.S. Pat. No. 5,384,253, incorporated herein byreference). Alternatively, recipient cells can be made more susceptibleto transformation by mechanical wounding.

Transfection of eukaryotic cells using electroporation has been quitesuccessful. Mouse pre-B lymphocytes have been transfected with humankappa-immunoglobulin genes (Potter et al., 1984), and rat hepatocyteshave been transfected with the chloramphenicol acetyltransferase gene(Tur-Kaspa et al., 1986) in this manner.

To effect transformation by electroporation in cells such as, forexample, plant cells, one may employ either friable tissues, such as asuspension culture of cells or embryogenic callus or alternatively onemay transform immature embryos or other organized tissue directly. Inthis technique, one would partially degrade the cell walls of the chosencells by exposing them to pectin-degrading enzymes (pectolyases) ormechanically wounding in a controlled manner Examples of some specieswhich have been transformed by electroporation of intact cells includemaize (U.S. Pat. No. 5,384,253; Rhodes et al., 1995; D'Halluin et al.,1992), wheat (Zhou et al., 1993), tomato (Hou and Lin, 1996), soybean(Christou et al., 1987) and tobacco (Lee et al., 1989).

One also may employ protoplasts for electroporation transformation ofplant cells (Bates, 1994; Lazzeri, 1995). For example, the generation oftransgenic soybean plants by electroporation of cotyledon-derivedprotoplasts is described by Dhir and Widholm in International PatentApplication No. WO 9217598, incorporated herein by reference. Otherexamples of species for which protoplast transformation has beendescribed include barley (Lazerri, 1995), sorghum (Battraw et al.,1991), maize (Bhattacharjee et al., 1997), wheat (He et al., 1994) andtomato (Tsukada, 1989).

4. Calcium Phosphate

In other embodiments of the present disclosure, a nucleic acid isintroduced to the cells using calcium phosphate precipitation. Human KBcells have been transfected with adenovirus 5 DNA (Graham and Van DerEb, 1973) using this technique. Also in this manner, mouse L(A9), mouseC127, CHO, CV-1, BHK, NIH3T3 and HeLa cells were transfected with aneomycin marker gene (Chen and Okayama, 1987), and rat hepatocytes weretransfected with a variety of marker genes (Rippe et al., 1990).

5. DEAE-Dextran

In another embodiment, a nucleic acid is delivered into a cell usingDEAE-dextran followed by polyethylene glycol. In this manner, reporterplasmids were introduced into mouse myeloma and erythroleukemia cells(Gopal, 1985).

6. Sonication Loading

Additional embodiments of the present disclosure include theintroduction of a nucleic acid by direct sonic loading. LTK-fibroblastshave been transfected with the thymidine kinase gene by sonicationloading (Fechheimer et al., 1987).

7. Liposome-Mediated Transfection

In a further embodiment of the disclosure, a nucleic acid may beentrapped in a lipid complex such as, for example, a liposome. Liposomesare vesicular structures characterized by a phospholipid bilayermembrane and an inner aqueous medium. Multilamellar liposomes havemultiple lipid layers separated by aqueous medium. They formspontaneously when phospholipids are suspended in an excess of aqueoussolution. The lipid components undergo self-rearrangement before theformation of closed structures and entrap water and dissolved solutesbetween the lipid bilayers (Ghosh and Bachhawat, 1991). Alsocontemplated is an nucleic acid complexed with Lipofectamine (Gibco BRL)or Superfect (Qiagen).

Liposome-mediated nucleic acid delivery and expression of foreign DNA invitro has been very successful (Nicolau and Sene, 1982; Fraley et al.,1979; Nicolau et al., 1987). The feasibility of liposome-mediateddelivery and expression of foreign DNA in cultured chick embryo, HeLaand hepatoma cells has also been demonstrated (Wong et al., 1980).

In certain embodiments of the disclosure, a liposome may be complexedwith a hemagglutinating virus (HVJ). This has been shown to facilitatefusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments, aliposome may be complexed or employed in conjunction with nuclearnon-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yetfurther embodiments, a liposome may be complexed or employed inconjunction with both HVJ and HMG-1. In other embodiments, a deliveryvehicle may comprise a ligand and a liposome.

8. Receptor Mediated Transfection

Still further, a nucleic acid may be delivered to a target cell viareceptor-mediated delivery vehicles. These take advantage of theselective uptake of macromolecules by receptor-mediated endocytosis thatwill be occurring in a target cell. In view of the cell type-specificdistribution of various receptors, this delivery method adds anotherdegree of specificity to the present disclosure.

Certain receptor-mediated gene targeting vehicles comprise a cellreceptor-specific ligand and a nucleic acid-binding agent. Otherscomprise a cell receptor-specific ligand to which the nucleic acid to bedelivered has been operatively attached. Several ligands have been usedfor receptor-mediated gene transfer (Wu and Wu, 1987; Wagner et al.,1990; Perales et al., 1994; Myers, EPO 0273085), which establishes theoperability of the technique. Specific delivery in the context ofanother mammalian cell type has been described (Wu and Wu, 1993;incorporated herein by reference). In certain aspects of the presentdisclosure, a ligand will be chosen to correspond to a receptorspecifically expressed on the target cell population.

In other embodiments, a nucleic acid delivery vehicle component of acell-specific nucleic acid targeting vehicle may comprise a specificbinding ligand in combination with a liposome. The nucleic acid(s) to bedelivered are housed within the liposome and the specific binding ligandis functionally incorporated into the liposome membrane. The liposomewill thus specifically bind to the receptor(s) of a target cell anddeliver the contents to a cell.

In still further embodiments, the nucleic acid delivery vehiclecomponent of a targeted delivery vehicle may be a liposome itself, whichwill preferably comprise one or more lipids or glycoproteins that directcell-specific binding. For example, lactosyl-ceramide, agalactose-terminal asialganglioside, have been incorporated intoliposomes and observed an increase in the uptake of the insulin gene byhepatocytes (Nicolau et al., 1987). It is contemplated that thetissue-specific transforming constructs of the present disclosure can bespecifically delivered into a target cell in a similar manner

9. Microprojectile Bombardment

Microprojectile bombardment techniques can be used to introduce anucleic acid into at least one, organelle, cell, tissue or organism(U.S. Pat. Nos. 5,550,318; 5,538,880; 5,610,042; and PCT Application WO94/09699; each of which is incorporated herein by reference). Thismethod depends on the ability to accelerate DNA-coated microprojectilesto a high velocity allowing them to pierce cell membranes and entercells without killing them (Klein et al., 1987). There are a widevariety of microprojectile bombardment techniques known in the art, manyof which are applicable to the disclosure.

Microprojectile bombardment may be used to transform various cell(s),tissue(s) or organism(s), such as for example any plant species.Examples of species which have been transformed by microprojectilebombardment include monocot species such as maize (PCT Application WO95/06128), barley (Ritala et al., 1994; Hensgens et al., 1993), wheat(U.S. Pat. No. 5,563,055, incorporated herein by reference), rice(Hensgens et al., 1993), oat (Torbet et al., 1995; Torbet et al., 1998),rye (Hensgens et al., 1993), sugarcane (Bower et al., 1992), and sorghum(Casas et al., 1993; Hagio et al., 1991); as well as a number of dicotsincluding tobacco (Tomes et al., 1990; Buising and Benbow, 1994),soybean (U.S. Pat. No. 5,322,783, incorporated herein by reference),sunflower (Knittel et al. 1994), peanut (Singsit et al., 1997), cotton(McCabe and Martinell, 1993), tomato (VanEck et al. 1995), and legumesin general (U.S. Pat. No. 5,563,055, incorporated herein by reference).

In this microprojectile bombardment, one or more particles may be coatedwith at least one nucleic acid and delivered into cells by a propellingforce. Several devices for accelerating small particles have beendeveloped. One such device relies on a high voltage discharge togenerate an electrical current, which in turn provides the motive force(Yang et al., 1990). The microprojectiles used have consisted ofbiologically inert substances such as tungsten or gold particles orbeads. Exemplary particles include those comprised of tungsten,platinum, and preferably, gold. It is contemplated that in someinstances DNA precipitation onto metal particles would not be necessaryfor DNA delivery to a recipient cell using microprojectile bombardment.However, it is contemplated that particles may contain DNA rather thanbe coated with DNA. DNA-coated particles may increase the level of DNAdelivery via particle bombardment but are not, in and of themselves,necessary.

For the bombardment, cells in suspension are concentrated on filters orsolid culture medium. Alternatively, immature embryos or other targetcells may be arranged on solid culture medium. The cells to be bombardedare positioned at an appropriate distance below the macroprojectilestopping plate.

An illustrative embodiment of a method for delivering DNA into a cell(e.g., a plant cell) by acceleration is the Biolistics Particle DeliverySystem, which can be used to propel particles coated with DNA or cellsthrough a screen, such as a stainless steel or Nytex screen, onto afilter surface covered with cells, such as for example, a monocot plantcells cultured in suspension. The screen disperses the particles so thatthey are not delivered to the recipient cells in large aggregates. It isbelieved that a screen intervening between the projectile apparatus andthe cells to be bombarded reduces the size of projectiles aggregate andmay contribute to a higher frequency of transformation by reducing thedamage inflicted on the recipient cells by projectiles that are toolarge.

V. Proteins, Polypeptides, and Peptides

In some cases, embodiments may utilize one or more purified cardiac cellprogramming factors, such as Hand2, myocardin, Gata4, Mef2c, or Tbx5proteins, polypeptides, or peptides, or one or more chromatingdestabilizing agent proteins, polypeptides, or peptides, or otherproteins, polypeptides, or peptides, and this may be done in addition toor alternative to utilizing the respective nucleic acid form. The term“purified proteins, polypeptides, or peptides” as used herein, isintended to refer to an proteinaceous composition, isolatable frommammalian cells or recombinant host cells, wherein the at least oneprotein, polypeptide, or peptide is purified to any degree relative toits naturally-obtainable state, i.e., relative to its purity within acellular extract. A purified protein, polypeptide, or peptide thereforealso refers to a wild-type or mutant protein, polypeptide, or peptidefree from the environment in which it naturally occurs.

The nucleotide and protein, polypeptide and peptide sequences forvarious genes have been previously disclosed, and may be found atcomputerized databases known to those of ordinary skill in the art. Onesuch database is the National Center for Biotechnology Information'sGenBank® and GenPept® databases. The coding regions for these knowngenes may be amplified and/or expressed using the techniques disclosedherein or by any technique that would be known to those of ordinaryskill in the art. Additionally, peptide sequences may be synthesized bymethods known to those of ordinary skill in the art, such as peptidesynthesis using automated peptide synthesis machines, such as thoseavailable from Applied Biosystems (Foster City, Calif.).

Generally, “purified” will refer to a specific protein, polypeptide, orpeptide composition that has been subjected to fractionation to removevarious other proteins, polypeptides, or peptides, and which compositionsubstantially retains its activity, as may be assessed, for example, bythe protein assays, as described herein below, or as would be known toone of ordinary skill in the art for the desired protein, polypeptide orpeptide.

Where the term “substantially purified” is used, this will refer to acomposition in which the specific protein, polypeptide, or peptide formsthe major component of the composition, such as constituting about 50%of the proteins in the composition or more. In preferred embodiments, asubstantially purified protein will constitute more than 60%, 70%, 80%,90%, 95%, 99% or even more of the proteins in the composition.

A peptide, polypeptide or protein that is “purified to homogeneity,” asapplied to the present disclosure, means that the peptide, polypeptideor protein has a level of purity where the peptide, polypeptide orprotein is substantially free from other proteins and biologicalcomponents. For example, a purified peptide, polypeptide or protein willoften be sufficiently free of other protein components so thatdegradative sequencing may be performed successfully.

Various methods for quantifying the degree of purification of proteins,polypeptides, or peptides will be known to those of skill in the art inlight of the present disclosure. These include, for example, determiningthe specific protein activity of a fraction, or assessing the number ofpolypeptides within a fraction by gel electrophoresis.

To purify a desired protein, polypeptide, or peptide a natural orrecombinant composition comprising at least some specific proteins,polypeptides, or peptides will be subjected to fractionation to removevarious other components from the composition. In addition to thosetechniques described in detail herein below, various other techniquessuitable for use in protein purification will be well known to those ofskill in the art. These include, for example, precipitation withammonium sulfate, PEG, antibodies and the like or by heat denaturation,followed by centrifugation; chromatography steps such as ion exchange,gel filtration, reverse phase, hydroxylapatite, lectin affinity andother affinity chromatography steps; isoelectric focusing; gelelectrophoresis; and combinations of such and other techniques.

Another example is the purification of a specific fusion protein using aspecific binding partner. Such purification methods are routine in theart. As the present disclosure provides DNA sequences for the specificproteins, any fusion protein purification method can now be practiced.This is exemplified by the generation of an specific protein-glutathioneS-transferase fusion protein, expression in E. coli, and isolation tohomogeneity using affinity chromatography on glutathione-agarose or thegeneration of a polyhistidine tag on the N- or C-terminus of theprotein, and subsequent purification using Ni-affinity chromatography.However, given many DNA and proteins are known, or may be identified andamplified using the methods described herein, any purification methodcan now be employed.

Although preferred for use in certain embodiments, there is no generalrequirement that the protein, polypeptide, or peptide always be providedin their most purified state. Indeed, it is contemplated that lesssubstantially purified protein, polypeptide or peptide, which arenonetheless enriched in the desired protein compositions, relative tothe natural state, will have utility in certain embodiments.

Methods exhibiting a lower degree of relative purification may haveadvantages in total recovery of protein product, or in maintaining theactivity of an expressed protein. Inactive products also have utility incertain embodiments, such as, e.g., in determining antigenicity viaantibody generation.

VI. Host Cells

Although in some embodiments the nucleic acids of the disclosure areprovided directly to cardiac tissue and are uptaken by cells in thetissue, in some embodiments the nucleic acids are first generated andmanipulated in cells ex vivo, such as by employing routine recombinanttechnology methods.

As used herein, the terms “cell,” “cell line,” and “cell culture” may beused interchangeably. All of these terms also include their progeny,which is any and all subsequent generations. It is understood that allprogeny may not be identical due to deliberate or inadvertent mutations.In the context of expressing a heterologous nucleic acid sequence, “hostcell” refers to a prokaryotic or eukaryotic cell, and it includes anytransformable organism that is capable of replicating a vector and/orexpressing a heterologous gene encoded by a vector. A host cell can, andhas been, used as a recipient for vectors. A host cell may be“transfected” or “transformed,” which refers to a process by whichexogenous nucleic acid is transferred or introduced into the host cell.A transformed cell includes the primary subject cell and its progeny. Asused herein, the terms “engineered” and “recombinant” cells or hostcells are intended to refer to a cell into which an exogenous nucleicacid sequence, such as, for example, a vector, has been introduced.Therefore, recombinant cells are distinguishable from naturallyoccurring cells which do not contain a recombinantly introduced nucleicacid.

In certain embodiments, it is contemplated that RNAs or proteinaceoussequences may be co-expressed with other selected RNAs or proteinaceoussequences in the same host cell. Co-expression may be achieved byco-transfecting the host cell with two or more distinct recombinantvectors. Alternatively, a single recombinant vector may be constructedto include multiple distinct coding regions for RNAs or DNAs (as anactive agent) or polypeptides (as an active agent), which could then beexpressed in host cells transfected with the single vector.

A tissue may comprise a host cell or cells to be transformed with apolynucleotide encoding part or all of p63, p53, and/or p21 shRNA orsiRNA, one or more cardiac cell reprogramming factors, and/or one ormore chromatin destabilizing agents. In specific embodiments, a cell mayharbor a polynucleotide encoding part or all of p63 shRNA, Hand2, and/ormyocardin. The tissue may be part or separated from an organism. Incertain embodiments, a tissue may comprise, but is not limited to,myocytes, adipocytes, alveolar, ameloblasts, axon, basal cells, blood(e.g., lymphocytes), blood vessel, bone, bone marrow, brain, breast,cardiac, cartilage, cervix, colon, cornea, embryonic, endometrium,endothelial, epithelial, esophagus, facia, fibroblast, follicular,ganglion cells, glial cells, goblet cells, kidney, liver, lung, lymphnode, muscle, neuron, ovaries, pancreas, peripheral blood, prostate,skin, skin, small intestine, spleen, stem cells, stomach, testes, and soforth.

In certain embodiments, the host cell or tissue may be comprised in atleast one organism. In certain embodiments, the organism may be, but isnot limited to, a prokayote (e.g., a eubacteria, an archaea) or aneukaryote, as would be understood by one of ordinary skill in the art(see, for example, webpagehttp://phylogeny.arizona.edu/tree/phylogeny.html).

Numerous cell lines and cultures are available for use as a host cell,and they can be obtained through the American Type Culture Collection(ATCC), which is an organization that serves as an archive for livingcultures and genetic materials (www.atcc.org). An appropriate host canbe determined by one of skill in the art based on the vector backboneand the desired result. A plasmid or cosmid, for example, can beintroduced into a prokaryote host cell for replication of many vectors.Cell types available for vector replication and/or expression include,but are not limited to, bacteria, such as E. coli (e.g., E. coli strainRR1, E. coli LE392, E. coli B, E. coli X 1776 (ATCC No. 31537) as wellas E. coli W3110 (F-, lambda-, prototrophic, ATCC No. 273325), DH5α,JM109, and KC8, bacilli such as Bacillus subtilis; and otherenterobacteriaceae such as Salmonella typhimurium, Serratia marcescens,various Pseudomonas specie, as well as a number of commerciallyavailable bacterial hosts such as SURE® Competent Cells and Solopack™Gold Cells (Stratagene®, La Jolla). In certain embodiments, bacterialcells such as E. coli LE392 are particularly contemplated as host cellsfor phage viruses.

Examples of eukaryotic host cells for replication and/or expression of avector include, but are not limited to, HeLa, NIH3T3, Jurkat, 293, Cos,CHO, Saos, and PC12. Many host cells from various cell types andorganisms are available and would be known to one of skill in the art.Similarly, a viral vector may be used in conjunction with either aeukaryotic or prokaryotic host cell, particularly one that is permissivefor replication or expression of the vector.

Some vectors may employ control sequences that allow it to be replicatedand/or expressed in both prokaryotic and eukaryotic cells. One of skillin the art would further understand the conditions under which toincubate all of the above described host cells to maintain them and topermit replication of a vector. Also understood and known are techniquesand conditions that would allow large-scale production of vectors, aswell as production of the nucleic acids encoded by vectors and theircognate polypeptides, proteins, or peptides.

VII. Combination Therapy

In certain cases, the therapy of the present disclosure is utilized inconjunction with one or more other therapies for a cardiac medicalcondition. p63, p53, and/or p21-inactivating agents may be used inconjunction with one or more cardiac cell reprogramming factors and/orin conjunction with one or more chromatin destabilizing agents and/orwith one or more anti-fibrotic agents or angiogenic factors. In specificembodiments, p63 shRNA or siRNA is used in combination with Hand2 and ormyocardin gene therapy, although it may also be used in combination withother genes or gene products, including, Gata4, Mef2c, Tbx5, miR-133,miR-1, Oct4, Klf4, c-myc, Sox2, Mesp1, Brachyury, Nkx2.5, ETS2, ESRRG,Mrtf-A, MyoD, and/or ZFPM2 (in nucleic acid or polypeptide or peptideform, in specific embodiments). The one or more other therapies may bedirectly or indirectly related to the cardiac medical condition(examples of indirectly related therapies include those for pain orinfection). In specific embodiments, the additional therapy related tothe cardiac medical condition is drug therapy, surgery, ventricularassisted device (VAD) implantation, video assisted thoracotomy (VAT),coronary bypass, or a combination thereof.

In specific embodiments, one or more agents that prevent fibrosis and/orenhance or promote angiogenesis may be used as adjuncts to embodimentsof the disclosure. They may be provided to an individual in a localizedregion of the heart, including a region that has tissue damage, loss ofcardiomyocyte, scar tissue, and so forth, or they may be providedsystemically. The one or more agents may be any composition suitable tofacilitate angiogenesis in the desired region. In specific embodiments,the agent may be a protein, peptide, small molecule, nucleic acid, andso forth. Embodiments such as those described in US2003/0103943 orUS2001/0041679 may be employed in conjunction with the methods of thedisclosure. Specific embodiments include fibroblast growth factor (FGF);vascular endothelial growth factor (VEGF); angiopoietins, Ang1 and Ang2;matrix metalloproteinase (MMP); Delta-like ligand 4 (DII4); or peptidesthereof; or combinations thereof. ITD-1 is a small molecule thatinhibits TGF-beta and thus, fibrosis and cardiac remodeling (Willems E,Cabral-Teixeira J, Schade D, et al. Cell Stem Cell. 2012. pp. 242-252),and it may be utilized.

The agent that enhances angiogenesis may be referred to as an angiogenicfactor. The agent may be provided to the individual prior to theindividual receiving the agent that partially or completelydownregulates or inactivates p63, p53, and/or 21 and/or prior to thecardiac cell reprogramming factor and/or prior to the chromatindestabilizing agent.

The therapy of the present disclosure may precede or follow the otheragent treatment by intervals ranging from minutes to hours to days toweeks or months. In embodiments where the other agent and the instanttherapy are applied separately to the individual, one would generallyensure that a significant period of time did not expire between the timeof each delivery, such that the therapy of the disclosure and theadditional therapy would still be able to exert an advantageouslycombined effect on the individual. In such instances, it is contemplatedthat one may contact the individual with both modalities simultaneouslyor within minutes of each other or within about 1-12, 6-12, or 12-24 hof each other. In some situations, it may be desirable to extend thetime period for treatment significantly, however, where several days (2,3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapsebetween the respective administrations.

In specific embodiments, the therapy of the present disclosure and theadditional therapy are provided at the same time or at different times.The separate entities may be within the same compositions or they may becomprised in separate compositions. In cases wherein the therapy of thepresent disclosure and the second therapy are provided at differenttimes, they may be separated by any suitable range in times, such asminutes, hours, days, or weeks. In embodiments wherein they are providedseparately, the order of delivery of two (or more) therapies may be ofany suitable order, including delivery of p63shRNA with Hand2 and/ormyocardin prior to or subsequent to another therapy.

Examples of other treatments to be employed with the therapy of thedisclosure includes one or more of the following: ACE Inhibitors,Aldosterone Inhibitor, Angiotensin II Receptor Blocker (ARBs);Beta-Blockers, Calcium Channel Blockers, Cholesterol-Lowering Drugs,Digoxin, Diuretics, Inotropic Therapy, Potassium or Magnesium,Vasodilators, anticoagulant medication, aspirin, surgery, VADimplantation, VAT, coronary bypass, percutaneous coronary intervention(PCI) or a combination thereof.

VIII. Kits of the Disclosure

Any of the compositions described herein may be comprised in a kit. In anon-limiting example, a p63, p53, and/or p21-inactivating agent (such asa siRNA or shRNA), one or more cardiac cell reprogramming factors,and/or one or more chromatin destabilizing agents or otherpolynucleotide or primers for amplification of same may be comprised ina kit. In specific embodiments, the kit comprises p63 shRNA with orwithout Hand2 and/or myocardin polypeptides or peptides. The kit mayadditionally comprise additional agents for therapy of a cardiac medicalcondition.

The components of the kits may be packaged either in aqueous media or inlyophilized form. The container means of the kits will generally includeat least one vial, test tube, flask, bottle, syringe or other containermeans, into which a component may be placed, and preferably, suitablyaliquoted. Where there are more than one component in the kit, the kitalso will generally contain a second, third or other additionalcontainer into which the additional components may be separately placed.However, various combinations of components may be comprised in a vial.The kits of the present disclosure also will typically include a meansfor containing the one or more compositions in close confinement forcommercial sale. Such containers may include injection or blow-moldedplastic containers into which the desired vials are retained.

The composition may be formulated into a syringeable composition. Inwhich case, the container means may itself be a syringe, pipette, and/orother such like apparatus, from which the formulation may be applied toan infected area of the body, injected into an animal, and/or evenapplied to and/or mixed with the other components of the kit. However,the components of the kit may be provided as dried powder(s). Whenreagents and/or components are provided as a dry powder, the powder canbe reconstituted by the addition of a suitable solvent. It is envisionedthat the solvent may also be provided in another container means.

The kits of the present disclosure will also typically include a meansfor containing the vials in close confinement for commercial sale, suchas, e.g., injection and/or blow-molded plastic containers into which thedesired vials are retained.

In particular embodiments, the kit comprises reagents and/or tools fordetermining that an individual has a cardiac medical condition. In someembodiments, the kit comprises one or more additional therapies for acardiac-related medical condition, such as one or more of ACE Inhibitor,aldosterone inhibitor, angiotensin II receptor blocker (ARBs);beta-blocker, calcium channel blocker, cholesterol-lowering drug,digoxin, diuretics, inotropic therapy, potassium, magnesium,vasodilator, anticoagulant medication, aspirin, TGF-beta inhibitor, anda combination thereof. In specific embodiments, an individual receivesangiogenic therapy before, during, or after the therapy of the presentdisclosure. Examples of angiogenic therapies include fibroblast growthfactor (FGF); vascular endothelial growth factor (VEGF); angiopoietins,Ang1 and Ang2; matrix metalloproteinase (MMP); Delta-like ligand 4(DII4); or peptides thereof; or combinations thereof.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the disclosure. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow presenttechniques discovered by the inventors to function well in the practiceof the disclosure, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe disclosure.

Example 1 P63 Inactivation Increases the Transdifferentiation Efficiencyof Fibroblasts into Cardiomyocytes

Cellular regenerative therapy has emerged as a treatment for a number ofdegenerative diseases, most notably ischemic heart disease, which is thenumber one cause of death in the world. Several strategies have beenproposed, including transdifferentiation and the formation of inducedpluripotent stem cells. Transdifferentiation has been attempted withtranscription factors such as Gata4, Mef2c and Tbx5 (GMT), while iPScells may be formed using the Yamanaka factors (Oct4, Sox2, Klf4, c-myc)or knocking down genes involved in regulating the cell cycle, such asp53.

While it has been reported that murine cells that are p63 deficientdisplay a high degree of plasticity, no distinct strategy has beendeveloped for their application in ischemic heart disease. Specifically,it has not been described how the p63 deficient cells may be “driven”down the cardiomyocyte lineage. The attached slides show FACS(fluorescence activated cell sorting) data confirming that 76% of p63deficient murine embryonic fibroblasts exposed to factors Hand2 andMyocardin express cTnT (Cardiac Troponin T), a highly specific markerfor the cardiomyocyte lineage. Without Hand2 and Myocardin,approximately 30-40% of p63−/− cells express cTnT while the remainder ofcells may have the potential to become other types of cells, which isnot ideal as a therapy. The strategy is unique because it selects forthe formation of induced cardiomyocytes over other lineages. Neitherthis strategy (inactivation of the p63 gene with the addition of Hand2and Myocardin) nor this high of an efficiency has been reported in theliterature. Approaches of the disclosure are useful clinical therapiesfor heart failure.

The p63 gene, a family member of the p53 gene, plays an important rolein cell cycle regulation, “stemness,” senescence, and differentiation.Wild type and p63 −/− mouse embryonic fibroblasts (MEF's) with andwithout exposure to lentiviral GMT (Gata4, Mef2c, Tbx5) were analyzed byflow cytometry for cTnT (marker of cardiomyocyte lineage) at 22 days.

FIG. 1 shows experimental design for analyzing fibroblasttransdifferentiation into cardiomyocytes in vitro. GMT refers to thecombination of Gata4, Mef2c, and Tbx5. GFP refer to green fluorescenceprotein. FIG. 2 shows FACS analysis of a positive control comprisingneonatal rat cardiomyoblasts. FIG. 3 demonstrates a negative controlusing wild-type cells given no virus, and in FIG. 4 the wild-type cellsare exposed to GFP whereas in FIG. 5 the wild-type cells were given GMT.FIG. 6 shows a negative control (secondary antibody only) for p63−/−mouse embryonic cells showing 1.7% cTNT as noise. In FIG. 7, the p63−/−cells were given no virus, whereas in FIG. 8 the p63−/− cells were givenGFP and stained with only the negative control secondary antibody and inFIG. 9 the p63−/− cells were given GFP. FIG. 10 shows FACS results forp63−/− cells given GMT and both antibodies. FIG. 11 illustrates allresults as a function of cTnT expression.

Thus, untreated p63 −/− MEF's expressed cTnT 45% of the time whilewild-type MEF's expressed cTnT 3% of the time. Untreated p63 −/− MEF'sdisplay a 15 fold increase in the expression of cTnT compared towild-type MEF's. Therefore, cells deficient in p63 are able to reprograminto cardiomyocyte-like cells at a highly efficient rate, suggestingthat it is feasible to efficiently reprogram endogenous cardiacfibroblasts from infarcted or disease myocardium into cardiomyocytes.

Example 2

P63 Inactivation and Addition of Hand2, Myocardin Increases Formation ofCTNT+ Cells from Murine Embryonic Fibroblasts

FIG. 12 illustrates an experimental design for analyzingtransdifferentiation of fibroblasts into cardiomyocytes in vitro. FIG.13 shows FACS data for unstained and stained wild-type MEFs (negativecontrols). FIG. 14 shows wild type MEFs treated with lentiviral GFP andlentiviral GMT. In FIG. 15, wild-type MEFs were given the noted factors(lentiviral Hand2 & Myocardin) and exposed to both antibodies. In FIG.16, shows a positive control, h9c2 neonatal rat cardiomyoblasts. FIG. 17shows the p63 −/− (which concerns the inactivation of both the TA andDelta isoforms of the p63 gene) MEFs where no factors were provided. Theleft side shows the control stained with only the secondary antibodywhile the right shows cells stained with both antibodies. FIG. 18demonstrates the double KO p63−/− MEFs where GFP (left side showsstaining only with secondary as a control while the right side showsstaining with both antibodies). In FIG. 19, the double KO p63−/− MEFswere given lentiviral GMT factors and both primary and secondaryantibodies.

Next, the factors Hand2 (related to cardiac morphogenesis, formation ofventricles, aortic arch) and myocardin (smooth muscle differentiation;cardiac muscle specific activator of Serum Response Factor (whichregulates cell cycle, growth, and differentiation)) were utilized withthe cells. In FIG. 20, p63−/− MEFs were exposed to lentiviral Hand2 andmyocardin in the presence or absence of miR-590. FIG. 21 illustratesdelta Np63−/− MEFs (knockout of only the DeltaN isoform) either with nofactor, GFP, or GMT in the presence of both primary and secondaryantibodies. For FIG. 22, TAp63−/− MEFs (knockout of only the TA isoform)were exposed to either no factor, GFP, or GMT in the presence of bothprimary and secondary antibodies. FIG. 23 summarizes an experiment,illustrating the percentage of cells expressing cTnT as a marker forcardiomyocyte lineage. FIG. 24 demonstrates the expression of certainpro-cardiogenic factors as measured by qPCR of p63 knockout MEFs(H/M=human Hand2/Myocardin lentivirus (SystemsBio).

Furthermore, given the targets that the inventors have identified, it isfeasible to use a viral vector, mRNA delivery, nanoparticle deliverysystem, etc., in order to create the desired effects. The examplediagram (FIG. 25) provided shows a lentiviral vector with p63 shRNA andtranscription factors Hand2 and Myocardin. Other similar approacheswould involve an adenoviral vector or the use of modified mRNA for Hand2and Myocardin combined with p63 siRNA oligonucleotides. Other methods ofsilencing p63 would involve the use of small molecule inhibitors orshRNA targeting other downstream regulatory genes. In summary, any ofthese approaches would allow for inactivation of the p63 gene with theaddition of factors Hand2 and Myocardin, resulting in the desiredeffect.

Example 3 P63 Inactivation and the Addition of Several Other Factors MayFurther Enhance the Transdifferentiation of Fibroblasts intoCardiomyocytes

Examples 1 and 2 illustrated two embodiments of applications of thisdisclosure. However, there are several other transcription factors thatmay be used to further enhance the efficiency: Gata4, Mef2c, Tbx5,miR-133, miR-1, Oct4, Klf4, c-myc, Sox2, Mesp1, Brachyury, Nkx2.5, ETS2,ESRRG, Mrtf-A, MyoD, ZFPM2 (in nucleic acid or polypeptide or peptideform, in specific embodiments). Adjunctive therapy could comprise ITD-1(TGF beta inhibitor), VEGF, surgery (coronary artery bypass), PCI, ormedial therapy, for example.

Example 4 Adjunct Therapy Comprising Downregulation of Snai1

In specific embodiments adjuncts to p63, p53, and/or p21 inactivationcomprise anti-fibrotic therapy, with or without the addition of cardiaccell reprogramming factors, chromatin destabilizing agents, and/orangiogenic therapy. FIG. 26 confirms that GMT-treated cells have lowerSnai1 expression, which plays a role in fibrosis. Snai1 (“Snail”) is adownstream effector of TGF-beta, which has been implicated in fibrosisor scar formation post-myocardial infarction. In specific embodiments,one can combine the reprogramming efficiency of p63, p53, and/or p21inactivation with one or more anti-fibrotic agents, such as ananti-Snai1 agent (for example, siRNA, antibody, small molecule such asITD-1, etc.) in order to regenerate at least part of the myocardiumafter a cardiac medical condition, such as a heart attack.

Example 5 P63 Knockdown in Mammalian Cells

FIG. 27 illustrates an example protocol of how p63 can be knocked downusing shRNA in mouse embryonic fibroblasts (MEFs) in vitro. Theefficiency of p63 knockdown in mouse embryonic fibroblasts (MEFs) isdemonstrated in FIG. 28. In the figure, it is shown by qPCR the p63knockdown efficiency in the MEFs using shRNA lentivirus as an example ofa vector. Puromycin selection (1 ug/mL) was performed after infection,and knockdown efficiency was measured by quantitative PCR 5-7 days afterselection. FIG. 28 demonstrates efficient knockdown of p63 using anexemplary p63 shRNA on a lentivirus vector. FIG. 29 demonstratesmaintenance of p63 knockdown at 3 weeks. In particular, qPCR data wasgathered at 3 weeks following infection of MEFs with p63 shRNAlentivirus. Outcome was measure as the fold change of knockdown relativeto non-silencing shRNA. The data shows that silencing p63 with shRNAresults in upregulation of cTnT and pro-cardiogenic factors Mef2c, Tbx5,Hand2 and Myocardin.

FIG. 30 demonstrates that p63 knockdown is efficient in other types ofcells than MEFs by demonstrating p63 knockdown in adult humankeratinocytes, or skin cells. According to the left panel, using qPCRthe p63 knockdown efficiency was determined in adult human keratinocytestreated with p63 shRNA. Thus, adult human keratinocytes may also bereprogrammed into cardiomyocyte-like cells using p63 knockdown withshRNA. The right panel in FIG. 30 shows by qPCR that there is increasedcardiac troponin T expression in adult human keratinocytes treated withp63 shRNA. p63 knockdown alone results in a 5-fold higher expression ofcTnT while p63 knockdown in addition to Hand2/Myocardin results in a20-fold higher expression of cTnT. Therefore, FIG. 30 provides data thatsilencing p63 works in adult human cells.

FIG. 31 demonstrates by qPCR that there is increased expression ofpro-cardiogenic factors and cardiac structural markers in adult humankeratinocytes treated with p63 shRNA. In particular, p63 knockdownenhances reprogramming via induction of Gata4, Mef2c, Tbx5, Hand2 andMyocardin. This parallels findings with p63 knockout MEFs which alsoexpressed these markers. p63 knockdown also leads to increasedexpression of cardiac structural markers MYH6, MYH7 and cardiacprogenitor markers Nkx2.5, c-kit, Is1-1 and Brachyury.

In specific embodiments, one or more p63, p53, and/or p21 inactivationagents are provided to an individual in need thereof and the individualalso receives one or more anti-fibrotic agents. In some cases theanti-fibrotic agent is provided to the individual before, during, and/orafter the one or more anti-fibrotic agents. In cases wherein theindividual also receives one or more cardiac cell reprogramming factorsand/or one or more chromatin destabilizing agents, the one or moreanti-fibrotic agent may be provided to the individual before, during,and/or after the respective factors or agents.

Although the present disclosure and its advantages have been describedin detail, it should be understood that various changes, substitutionsand alterations can be made herein without departing from the spirit andscope of the disclosure as defined by the appended claims. Moreover, thescope of the present application is not intended to be limited to theparticular embodiments of the process, machine, manufacture, compositionof matter, means, methods and steps described in the specification. Asone of ordinary skill in the art will readily appreciate from thedisclosure of the present disclosure, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized according to the present disclosure.Accordingly, the appended claims are intended to include within theirscope such processes, machines, manufacture, compositions of matter,means, methods, or steps.

What is claimed is:
 1. A method of in vivo reprogramming ofdifferentiated cardiac cells, comprising the step of providing atherapeutically effective amount of one or more compositions to theheart of an individual, wherein said one or more compositions consistsessentially of (1) an agent that partially or completely downregulatesor inactivates p63, (2) Hand2 nucleic acid or polypeptide, and (3)myocardin nucleic acid or polypeptide, wherein said differentiatedcardiac cells are reprogrammed to cardiomyocyte-like cells.
 2. Themethod of claim 1, wherein the agent partially or completelydownregulates or inactivates expression of p63.
 3. The method of claim2, wherein the agent comprises p63 shRNA or siRNA.
 4. The method ofclaim 1, further comprising the step of providing to the individual aneffective amount of one or more cardiac cell reprogramming factors. 5.The method of claim 4, wherein the one or more cardiac cellreprogramming factors is a polypeptide, peptide, or nucleic acid.
 6. Themethod of claim 4, wherein the one or more cardiac cell reprogrammingfactors is Gata4, Mef2c, Tbx5, Mesoderm posterior protein 1 (Mesp1),miR-133, miR-1, Oct4, Klf4, c-myc, Sox2, Brachyury, Nkx2.5, ETS2, ESRRG,Mrtf-A, MyoD, ZFPM2, or a combination thereof.
 7. The method of claim 1,wherein the one or both of Hand2 and myocardin nucleic acids orpolypeptides are in the same composition as the agent that inactivatesp63.
 8. The method of claim 1, wherein the one or both of Hand2 andmyocardin nucleic acids or polypeptides are in a different compositionas the agent that inactivates p63.
 9. The method of claim 4, wherein theagent that inactivates p63 is provided before the one or more cardiaccell reprogramming factors.
 10. The method of claim 1, wherein aneffective amount of one or more chromatin destabilizing agents areprovided to the individual.
 11. The method of claim 10, wherein the oneor more chromatin destabilizing agents are selected from the groupconsisting of Oct4, DZNep, Sall4, SOX2, KLF4, MYC, SB431542, PD0325901,Parnate, CHIR99021, A-83-01, NaB, PS48, Forskolin (FSK),2-methyl-5-hydroxytryptamine (2-Me-5HT), D4476, VPA, CHIR99021 (CHIR),616452, Tranylcypromine, Prostaglandin E2, Rolipram, 3-deazaneplanocin A(DZNep), 5-Azacytidine, sodium butyrate, RG108, and a combinationthereof.
 12. The method of claim 10, wherein the one or more chromatindestabilizing agents are provided to the individual prior to when theagent that inactivates p63 is provided to the individual.
 13. The methodof claim 10, wherein the one or more chromatin destabilizing agents areprovided to the individual prior to when the agent that inactivates p63is provided to the individual, and wherein the agent that inactivatesp63 is provided to the individual prior to when the one or more cardiaccell reprogramming factors are provided to the individual.
 14. Themethod of claim 1, wherein the differentiated cardiac cells arefibroblasts, endothelial cells, or a combination thereof.
 15. The methodof claim 1, wherein the agent that inactivates p63 comprises a nucleicacid and said nucleic acid is comprised on one or more vectors.
 16. Themethod of claim 4, wherein the one or more cardiac cell reprogrammingfactors comprise a nucleic acid and said nucleic acid is comprised onone or more vectors.
 17. The method of claim 10, wherein the one or morechromatin destabilizing agents comprise a nucleic acid and said nucleicacid is comprised on one or more vectors.
 18. The method of claim 15,wherein the nucleic acids are comprised on separate vectors.
 19. Themethod of claim 15, wherein the nucleic acids are comprised on the samevector.
 20. The method of claim 18, wherein the vector is a viral vectoror a non-viral vector.
 21. The method of claim 20, wherein the non-viralvector is a nanoparticle, plasmid, liposome, or a combination thereof.22. The method of claim 20, wherein the viral vector is an adenoviral,lentiviral, retroviral, or adeno-associated viral vector.
 23. The methodof claim 20, wherein p63 shRNA, Hand2, and/or myocardin nucleic acidsare comprised on a lentiviral vector.
 24. The method of claim 20,wherein the p63 shRNA, Hand2, and/or myocardin nucleic acids arecomprised on an adenoviral vector.
 25. The method of claim 1, whereinthe p63 comprises a nucleic acid and said nucleic acid is a modifiedmRNA molecule.
 26. The method of claim 4, wherein the one or morecardiac cell reprogramming factors comprises a nucleic acid and saidnucleic acid is a modified mRNA molecule.
 27. The method of claim 10,wherein the one or more chromatin destabilizing agents comprises anucleic acid and said nucleic acid is a modified mRNA molecule.
 28. Themethod of claim 1, further comprising the step of delivering to theindividual an additional cardiac therapy.
 29. The method of claim 28,wherein the additional cardiac therapy comprises drug therapy, surgery,ventricular assist device (VAD) implantation, video assisted thoracotomy(VAT) coronary bypass, percutaneous coronary intervention (PCI), or acombination thereof.
 30. The method of claim 1, wherein the cardiac cellis a dividing cell or a non-dividing cell.
 31. The method of claim 15,wherein a promoter on the vector is a cell-specific promoter.
 32. Themethod of claim 15, wherein a promoter on the vector is afibroblast-specific promoter.
 33. The method of claim 1, wherein theagent that inactivates p63 inactivates the TA isoform of p63.
 34. Themethod of claim 1, wherein the providing step is further defined asinjecting the agent into the heart.
 35. The method of claim 4, whereinthe providing step is further defined as injecting the one or morecardiac cell reprogramming factors into the heart.
 36. The method ofclaim 10, wherein the providing step is further defined as injecting theone or more chromatin stabilizing agents into the heart.
 37. The methodof claim 1, further comprising the step of providing one or moreanti-fibrotic agents.
 38. The method of claim 37, wherein the one ormore anti-fibrotic agents comprises at least one anti-Snail agent. 39.The method of claim 38, wherein the anti-Snail agent is a siRNA, shRNA,antibody, or small molecule.
 40. The method of claim 39, wherein thesmall molecule is ITD-1.